TLC-CLIP

Christina Ernst; Julien Duc; Didier Trono

May 10, 2023

TLC-CLIP

Nucleic Acids Research

DOI

dx.doi.org/10.17504/protocols.io.rm7vzywr4lx1/v1

Christina Ernst¹,
Julien Duc¹,
Didier Trono¹

¹EPFL - EPF Lausanne

Christina Ernst

EPFL - EPF Lausanne

DOI: dx.doi.org/10.17504/protocols.io.rm7vzywr4lx1/v1

External link: https://doi.org/10.1093/nar/gkad466

Protocol Citation: Christina Ernst, Julien Duc, Didier Trono 2023. TLC-CLIP . protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzywr4lx1/v1

Manuscript citation:

Ernst C, Duc J, Trono D (2023) Efficient and sensitive profiling of RNA–protein interactions using TLC-CLIP. Nucleic Acids Research 51(13). doi: 10.1093/nar/gkad466

License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Protocol status: Working

We use this protocol and it's working

Created: August 22, 2022

Last Modified: May 10, 2023

Protocol Integer ID: 68998

Keywords: RNA-Protein interactions, RNA library preparation, Crosslinking and Immunoprecipitation, CLIP, RNA-binding proteins, RBPs

Funders Acknowledgement:

Swiss National Science Foundation

Grant ID: PZ00P3_202048

Human Frontier Science Program

Grant ID: LT000147/2019

Abstract

RNA-binding proteins are instrumental for post-transcriptional gene regulation, controlling all aspects throughout the lifecycle of RNA molecules. However, transcriptome-wide methods to profile RNA-protein interactions in vivo remain technically challenging and require large amounts of starting material. Herein, we present an improved library preparation strategy for crosslinking and immunoprecipitation (CLIP) that is based on tailing and ligation of cDNA molecules (TLC). TLC involves the generation of solid-phase cDNA, followed by ribotailing to increase the efficiency of subsequent adapter ligation. These modifications result in a streamlined, fully bead-based library preparation strategy, which eliminates time-consuming purification procedures and drastically reduces sample loss, allowing the profiling of RNA-protein interactions from as few as 1000 cells.
In the accompanying manuscript, we have applied TLC-CLIP to four endogenous RNA-binding proteins, demonstrating its reproducibility and improved precision due to a higher number of crosslinking-induced deletions that serve as an intrinsic quality metric and increase both specificity and nucleotide-resolution.

Guidelines

While working with RNA during the inital parts of the protocol, keep samples cold (on ice) and in RNase-free environment. Use RNase-free water for reactions and buffers. 

Materials

Equipment
Equipment
UVP Crosslinker CL-3000
NAME
Analytic Jena
BRAND
UVPA849-95-0615-02
SKU
https://www.uvp.com/products/lab-equipment/uvp-crosslinker/
LINK

Equipment
Branson Tip Sonicator
NAME
Sonicator
TYPE
Branson
BRAND
LPe 40:0.50:4T
SKU

Equipment
ThermoMixer® C
NAME
Eppendorf
BRAND
Catalog No. 2231000680
SKU
https://online-shop.eppendorf.us/US-en/Temperature-Control-and-Mixing-44518/Instruments-44519/Eppendorf-ThermoMixerC-PF-19703.html
LINK

Equipment
SureLock™ Tandem Midi Gel Tank
NAME
Electrophoresis System
TYPE
Invitrogen
BRAND
STM1001
SKU
https://www.thermofisher.com/order/catalog/product/STM1001
LINK

Equipment
Criterion Blotter with Plate Electrodes
NAME
Wet-transfer system
TYPE
Bio-Rad
BRAND
1704070
SKU
https://www.bio-rad.com/en-ch/product/criterion-blotter?ID=351faa8e-680e-4892-9778-c8c722e9b3b2
LINK

Equipment
Odyssey CLx
NAME
Imaging System
TYPE
LI-COR
BRAND
Odyssey CLx
SKU
https://www.licor.com/bio/odyssey-clx/
LINK

Equipment
Bioanalyzer
NAME
Bioanalyzer
TYPE
Agilent
BRAND
G2991AA
SKU
https://www.agilent.com/en/product/bioanalyzer-automated-electrophoresis/bioanalyzer-instrument/2100-bioanalyzer-instrument-228250
LINK
Any bioanalyzer will suffice.
SPECIFICATIONS

Equipment
Qubit Fluorometer
NAME
Fluorometer
TYPE
Invitrogen
BRAND
Q33238
SKU
https://www.thermofisher.com/order/catalog/product/Q33238#/Q33238
LINK

Buffer and Stock Solutions
Reagent1M Tris-HCl pH=7.5Invitrogen - Thermo FisherCatalog #15567-027  
Reagent5M NaClAmbionCatalog #AM9760G  
ReagentIgepalMerck MilliporeSigma (Sigma-Aldrich)Catalog #I8896  
ReagentSDS, 10% SolutionLife TechnologiesCatalog #AM9822  
ReagentMagnesium Chloride Solution BioUltraMerck MilliporeSigma (Sigma-Aldrich)Catalog #68475-100ML-F  
ReagentTween 20Merck MilliporeSigma (Sigma-Aldrich)Catalog #P1379-500ml  
ReagentPEG400Merck MilliporeSigma (Sigma-Aldrich)Catalog #91893  
ReagentLithium chloride (8M)Merck MilliporeSigma (Sigma-Aldrich)Catalog #L7026-100ML  
ReagentEDTA (0.5 M), pH 8.0Life TechnologiesCatalog #AM9260G  
ReagentUltraPure&trade; DNase/RNase-Free Distilled WaterThermo FisherCatalog #10977049  

Chemicals
ReagentSodium deoxycholate (SDC)Merck MilliporeSigma (Sigma-Aldrich)Catalog #30970  
ReagentDTT, 1MThermo FisherCatalog #P2325  
ReagentMethanolFisher ScientificCatalog #code356T  
ReagentLithium dodecyl sulfateMerck MilliporeSigma (Sigma-Aldrich)Catalog #L4632  

Commercial Kits
ReagentPierce&trade; Rapid Gold BCA Protein Assay KitThermo FisherCatalog #A53225  
ReagentProNex® Size-Selective Purification SystemPromegaCatalog #NG2002  
ReagentBioAnalyzer High Sensitivity Chip Agilent TechnologiesCatalog #5067-4626  
ReagentQubit® dsDNA HS Assay KitThermo Fisher ScientificCatalog #Q32854  

Reagents
ReagentDynabeads&trade; Protein G for ImmunoprecipitationThermo FisherCatalog #10004D  
ReagentcOmplete™, Mini, EDTA-free (Protease Inhibitor)RocheCatalog ##11836170001)  
ReagentOligo(dT)25 DynabeadsThermo Fisher ScientificCatalog #61005  
ReagentPhusion HF Buffer PackThermo FisherCatalog #F518L 
ReagentPhusion High-Fidelity PCR Master Mix with HF Buffer - 500 rxns (50 ul vol)New England BiolabsCatalog #M0531L   

Enzymes
ReagentRNase I (10 U/&micro;L)Thermo FisherCatalog #EN0602  
ReagentTURBO™ DNase (2 U/µL)Thermo Fisher ScientificCatalog #AM2238  
ReagentSUPERaseIN RNase InhibitorThermo Fisher ScientificCatalog #AM2696  
ReagentT4 Polynucleotide Kinase - 2,500 unitsNew England BiolabsCatalog #M0201L  
ReagentT4 RNA Ligase 1 (ssRNA Ligase) - 5,000 unitsNew England BiolabsCatalog #M0204L  
ReagentProteinase K Solution (20 mg/mL)Thermo Fisher ScientificCatalog #AM2546  
ReagentSuperScript™ IV Reverse TranscriptaseThermo Fisher ScientificCatalog #18090050  
ReagentTerminal Deoxynucleotidyl TransferaseTakara Bio Inc.Catalog #2230B  
ReagentT4 RNA Ligase High ConcentrationNew England BiolabsCatalog #M0437   

SDS-PAGE
ReagentNuPAGE™ LDS Sample Buffer (4X)Invitrogen - Thermo FisherCatalog #NP0008  
Reagent2-mercaptoethanolMerck MilliporeSigma (Sigma-Aldrich)Catalog #M6250  
ReagentNuPAGE&trade; 4-12% Bis-Tris Midi Protein Gels, 20-well, w/adaptersThermo FisherCatalog #WG1402A  
ReagentNuPAGE™ MOPS SDS Running Buffer (20X)Invitrogen - Thermo FisherCatalog #NP000102  
ReagentNitrocellulose Membrane 0.45 umBio-Rad LaboratoriesCatalog #1620115  
ReagentNuPAGE&trade; Transfer Buffer (20X)Thermo FisherCatalog #NP00061  
ReagentPrecision Plus Protein All Blue Prestained Protein StandardsBio-Rad LaboratoriesCatalog #1610373  
ReagentThick Blot Filter Paper Precut 9.5 x 15.2 cmBio-Rad LaboratoriesCatalog #1704085  

Consumables
Reagent1.5 mL LoBind tubes EppendorfCatalog #022431021  
ReagentBRAND(TM) PCR TUBE STRIPS OF 8 ATTACHED SINGLE CAPS STANDARD PROFILE 0.2 MLMerck MilliporeSigma (Sigma-Aldrich)Catalog #BR781332-120EA  
ReagentRNaseZap®Thermo ScientificCatalog #AM9780  
ReagentPCR SealsThermo ScientificCatalog #AB0558  
ReagentSwann-Morton™ Sterile Disposable Stainless Steel ScalpelsFisher ScientificCatalog #11798343  
 
Oligonucleotides
NameSequenceScalePurificationIndex
3' Adapter
TLC-L3/5Phos/AGATCGGAAGAGCACACGTCTGAAAAAAAAAAAAAAAAAAAAAAAAA/3IR800CWN/250nmRNASE
5' Adapter
TLC_L01/5Phos/NNNNATCACGNNNNNAGATCGGAAGAGCGTCGTG/3ddC/100nmSTDCGTGAT
TLC_L02/5Phos/NNNNCGATGTNNNNNAGATCGGAAGAGCGTCGTG/3ddC/100nmSTDACATCG
TLC_L03/5Phos/NNNNTTAGGCNNNNNAGATCGGAAGAGCGTCGTG/3ddC/100nmSTDGCCTAA
TLC_L04/5Phos/NNNNTGACCANNNNNAGATCGGAAGAGCGTCGTG/3ddC/100nmSTDTGGTCA
TLC_L05/5Phos/NNNNACAGTGNNNNNAGATCGGAAGAGCGTCGTG/3ddC/100nmSTDCACTGT
TLC_L06/5Phos/NNNNGCCAATNNNNNAGATCGGAAGAGCGTCGTG/3ddC/100nmSTDATTGGC
TLC_L07/5Phos/NNNNCAGATCNNNNNAGATCGGAAGAGCGTCGTG/3ddC/100nmSTDGATCTG
TLC_L08/5Phos/NNNNACTTGANNNNNAGATCGGAAGAGCGTCGTG/3ddC/100nmSTDTCAAGT
TLC_L09/5Phos/NNNNGATCAGNNNNNAGATCGGAAGAGCGTCGTG/3ddC/100nmSTDCTGATC
TLC_L10/5Phos/NNNNTAGCTTNNNNNAGATCGGAAGAGCGTCGTG/3ddC/100nmSTDAAGCTA
TLC_L11/5Phos/NNNNATGAGCNNNNNAGATCGGAAGAGCGTCGTG/3ddC/100nmSTDGCTCAT
TLC_L12/5Phos/NNNNCTTGTANNNNNAGATCGGAAGAGCGTCGTG/3ddC/100nmSTDTACAAG
cDNA Amplification
P5short_TLC-CLIPACACGACGCTCTTCCGATCT100nmPAGE
P7short_TLC-CLIPTGACGTGTGCTCTTCCGATCT100nmPAGE
PCR amplification
P5_Universal_adapterAATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT1umPAGE
P7-1_TLC-CLIPCAAGCAGAAGACGGCATACGAGATCGTGATGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC*T250nmPAGEATCACG
P7-2_TLC-CLIPCAAGCAGAAGACGGCATACGAGATACATCGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC*T250nmPAGECGATGTAT
P7-3_TLC-CLIPCAAGCAGAAGACGGCATACGAGATGCCTAAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC*T250nmPAGETTAGGCAT
P7-4_TLC-CLIPCAAGCAGAAGACGGCATACGAGATTGGTCAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC*T250nmPAGETGACCAAT
P7-5_TLC-CLIPCAAGCAGAAGACGGCATACGAGATCACTGTGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC*T250nmPAGEACAGTGAT
P7-6_TLC-CLIPCAAGCAGAAGACGGCATACGAGATATTGGCGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC*T250nmPAGEGCCAATAT
P7-7_TLC-CLIPCAAGCAGAAGACGGCATACGAGATGATCTGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC*T250nmPAGECAGATCAT
P7-8_TLC-CLIPCAAGCAGAAGACGGCATACGAGATTCAAGTGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC*T250nmPAGEACTTGAAT
 

Safety warnings

Prolonged exposure to UVC light can cause skin and eye damage. Ensure that the UV-crosslinker functions properly and follow all safety precautions specified in the manufacturer's instructions.
Some of the chemicals and reagents used in this protocol can be hazardous if not handled properly. Always follow the safety precautions outlined in the Safety Data Sheets (SDS) provided by the manufacturer. 

Before start

Prepare Buffers

iCLIP Lysis Buffer
Final ConcentrationStock SolutionFor 500 ml
50 mM Tris-HCl, pH 7.41 M Tris-HCl, pH 7.525 ml
100 mM NaCl5 M NaCl 10 ml
1% Igepal-CA-630100% Igepal CA-6305 ml
0.1% SDS10% SDS5 ml
0.5% sodium deoxycholate2.5 g
Add H2O455 ml

High Salt Wash Buffer

Final ConcentrationStock SolutionFor 500 ml
50 mM Tris-HCl, pH 7.41 M Tris-HCl, pH 7.525 ml
1 M NaCl5 M NaCl 100 ml
1 mM EDTA0.5M EDTA, pH 8.01 ml
1% Igepal-CA-630100% Igepal CA-6305 ml
0.1% SDS10% SDS5 ml
0.5% sodium deoxycholate2.5 g
Add H2O364 ml


PNK Wash Buffer

Final ConcentrationStock SolutionFor 500 ml
20 mM Tris-HCl, pH 7.41 M Tris-HCl, pH 7.510 ml
10 mM MgCl22 M MgCl22.5 ml
0.2% Tween-20100% Tween-201 ml
Add H2O486.5 ml

5X PNK Buffer

Final ConcentrationStock SolutionFor 10 ml
350 mM Tris-HCl, pH 6.51 M Tris-HCl, pH 6.53.5 ml
50 mM MgCl22 M MgCl20.25 ml
5 mM DTT1M DTT0.05 ml
Add H2O6.2 ml
Freeze individual use aliquots to avoid freeze-thaw cycles. 

4X Ligation Buffer

Final ConcentrationStock SolutionFor 10 ml
200 mM Tris-HCl, pH 7.81 M Tris-HCl, pH 7.82 ml
40 mM MgCl22 M MgCl20.2 ml
4 mM DTT1M DTT0.04 ml
Add H2O7.76 ml
Freeze individual use aliquots to avoid freeze-thaw cycles. 


Proteinase K Buffer

Final ConcentrationStock SolutionFor 50 ml
100 mM Tris-HCl, pH 7.41 M Tris-HCl, pH 7.45 ml
50 mM LiCl8 M LiCl0.3125 ml
1 mM EDTA0.5 M EDTA0.1 ml
0.2% LiDS1% LiDS10 ml
Add H2O34.5875 ml

Oligo(dT) Binding Buffer

Final ConcentrationStock SolutionFor 50 ml
20 mM Tris-HCl, pH 7.41 M Tris-HCl, pH 7.41 ml
1 M LiCl8 M LiCl6.25 ml
2 mM EDTA0.5 M EDTA0.2 ml
Add H2O42.55 ml


Oligo(dT) Wash Buffer

Final ConcentrationStock SolutionFor 50 ml
10 mM Tris-HCl, pH 7.41 M Tris-HCl, pH 7.40.5 ml
150 mM LiCl8 M LiCl0.9375 ml
0.1 mM EDTA0.5 M EDTA0.01 ml
Add H2O48.5525 ml

First-Strand (FS) Buffer (5X)

Final ConcentrationStock SolutionFor 10 ml
250 mM Tris-HCl, pH 8.31 M Tris-HCl, pH 8.32.5 ml
375 mM KCl1 M KCl3.75 ml
15 mM MgCl21 M MgCl20.15 ml
Add H2O3.6 ml
 

Generation of preadenylated TLC-L3 adapter

TLC-L3 oligo was ordered from IDT at 250 nmole scale, carrying a 5' phosphorylation and 3' IRDye® 800CW (NHS Ester) (v3) modification and purified using RNase-free HPLC with a total yield of 21.1 nmoles. 

Set up 50 µl of 100 µM TLC-L3 adapter (5 nmoles) with 25 µl 10X 5' DNA Adenylation Reaction Buffer, 25 µl 1 mM ATP and 50 µl Mth RNA Ligase (1nmol) in a total volume of 200 µl using theReagent5’ DNA Adenylation Kit - 50 rxnsNew England BiolabsCatalog #E2610L . 
Incubate at 65ºC for 2 hours followed by inactivation at 85ºC for 10 minutes (reaction turns cloudy). 
Clean up using the ReagentNucleotide Removal KitQiagenCatalog #28304  by mixing the 200 µl preadenylation reaction with 4.8 ml PNI buffer and distributing over 10 columns of Nucleotide Removal Kit. 
Spin down at 6000 rpm for 30 seconds.
Wash once in 750 µl PE and spin for 1 minute at 6000 rpm, followed by an empty spin at full speed. 
Transfer to a new collection tube and add 50 µl H2O per column and incubate at RT for 2 minutes
Spin at 6000 rpm for 1 minute to elute
Combine eluates at an approximate final concentration of 10 µM and prepare 1 µM working stocks to be stored at -20ºC. 

UV crosslinking and generation of cell lysates

45m

UV crosslinking and generation of cell lysates

Grow desired cell line to ~80% confluency in appropriate culture conditions.

Remove media, wash once in ice-cold PBS and drain cells of all fluid.

Transfer the plate onto ice, remove lid of the culture dish, and crosslink at 254 nm with 300 mJ/cm2. 
TemperatureOn ice   
Equipment
UVP Crosslinker CL-3000
NAME
Analytic Jena
BRAND
UVPA849-95-0615-02
SKU
https://www.uvp.com/products/lab-equipment/uvp-crosslinker/
LINK

Scrape cells into 5 ml PBS, count then aliquot desired number of cells and spin down.

10m

Resuspend cell pellet in iCLIP Lysis Buffer - 1 ml buffer for 1 million cells - scale accordingly.

Place lysates on ice for 5 minutes.
TemperatureOn ice   Duration00:05:00   

Sonicate lysates for 10-20 seconds at 0.5sec ON and 0.5sec OFF at 10% amplitude to reduce viscosity. 

Temperature-80 °C Safe Stopping Point  

Quantify cell lysates with Pierce BCA Quantification Assay or other method of choice to determine protein concentration. 

Expected result
We aim for lysate concentrations of 0.5 µg/µl for CLIP experiments.

Concentrations will differ between samples and cell types - adjust the amount of iCLIP Lysis Buffer used in step 1.4 to your samples to reach the desired concentration. 

10m

Preparation of bead-antibody mixture

45m

Preparation of bead-antibody mixture
We routinely use 1-2 µg of antibody per IP in 25-50 µg of cell lysate. 
Note
100 µl of protein-G beads bind 20-30 µg of antibody - calculate total volume of beads necessary based on amount of antibody used for experiment. 

Wash appropriate amount of beads twice in 1 ml iCLIP Lysis Buffer, then resuspend in 100 µl per antibody

Add antibody to beads and incubate at room temperature for 30-60 minutes rotating. 

TemperatureRoom temperature   
Duration00:30:00   
Note
Continue with section 3 'RNase and DNase Treatment of Lysates' during this incubation step.

30m

Wash beads twice in 1 ml iCLIP Lysis buffer then resuspend in 10 µl 6X Protease Inhibitor per IP.

Note
Add the appropriate amount of antibody-bead mixture to cell lysate to achieve 1X Protease Inhibitor concentration.

RNase and DNase Treatment of Lysates

30m

RNase and DNase Treatment of Lysates
Perform these steps while beads are coupling to antibodies. 
Note
Ensure to use the same concentration and the same volume of lysate for a given RBP after optimisation of RNase concentrations.

When optimising RNase concentrations, starting points can be a final amount of 0.25U, 0.025U and 0.005U of RNaseI (EN0602) for 50.000 293T cells (50 µl of lysate at ~0.5 µg/µl).

Note
We aim for a lysate concentration of ~0.5 µg/µl, more concentrated lysates can lead to higher background signal, but can be necessary for lowly expressed RBPs. 

Make serial dilutions to achieve desired RNaseI dilution then add 10 µl of RNaseI and 2 µl of Turbo DNase to cell lysates

Digest RNA for exactly 3 minutes at 37°C shaking at 1100 rpm then immediately transfer to ice and incubate for another 3 minutes. 

Note
Keep digestion time consistent between experiments to avoid over-digestion of RNA. 

10m

Spin lysates for 10 minutes at 4°C at 16,000g then transfer lysates to a fresh tube. 
 Temperature4 °C   Duration00:10:00   

15m

Immunoprecipitation and Washes

2h 30m

Immunoprecipitation and Washes
Keep the timing of IP consistent between experiments as RNaseI remains in samples and has residual activity even at 4°C. 

Set up 50 µl of RNase-treated lysate with 10 µl of antibody-bead mixture in 6X Protease Inhibitor and incubate for 2 hours at 4°C. 
Temperature4 °C   Duration02:00:00   

Magnetically attract beads and remove supernatant.

Note
Unbound fraction of IP can be kept to test IP efficiency via Western Blot when optimising conditions. 

Wash 2 x in 200 µl High Salt Buffer and keep the second wash for at least 1 minute at 4°C.

10m

Wash 2 x in 200 µl PNK Wash Buffer and keep in PNK Wash Buffer until ready to proceed. 

10m

Dephosphorylation and first adapter ligation

45m

Dephosphorylation and first adapter ligation 

Prepare PNK reaction and add 20 µl per sample. 

PNK Reactionx 1x #
5X PNK Buffer4 µl
SuperaseIN0.5 µl
T4 PNK0.5 µl
H2O15 µl

10m

Incubate at 37°C for 20 minutes with interval mixing in ThermoMixer.
Shaker1800 rpm, 37°C 15 seconds shaking every 2 minutes  
Equipment
Eppendorf Thermomixer C Model 5382
NAME
Thermomixer C
TYPE
Eppendorf
BRAND
5382000023
SKU

20m

Remove PNK reaction and wash once in 200 µl PNK Wash Buffer. 

Prepare Ligation mix and add 20 µl per sample. 

Ligation Mixx 1x #
4X Ligation Buffer5 µl
T4 RNA Ligase1 µl
1 µM L3 Adapter1 µl
SuperaseIN0.5 µl
H2O8.5 µl
PEG4004 µl
Prepare Ligation Reaction without PEG, mix then add PEG400 and mix by pipetting 10X with P1000. 

10m

Incubate at 16°C overnight or at 25°C for 75 minutes with interval mixing in ThermoMixer. 

Shaker1800 rpm, 16°C 15 seconds shaking every 2 minutes  DurationOvernight   

Purification of RNA-protein complexes

4h 30m

Purification of RNA-protein complexes
Purification via SDS-PAGE can be omitted to enable a 2-day workflow (see "Omission of PAGE purification"). Please refer to the critical discussion of this step in Ernst et al. (2023) before choosing this workflow. 

Remove ligation reaction and wash twice in 200 µl High Salt Buffer and twice in 200 µl PNK Wash Buffer. 

Note
When omitting PAGE purification, see step case "noPAGE" for how to proceed. 

20m

Resuspend in 20 µl 1X LDS sample buffer containing 5% ß-mercapto-ethanol. 

Denature at 70°C for 1 minute then proceed to PAGE purification. 

Resolve RNA-protein complexes on NuPAGE 4-12% Bis-Tris Gel at 180V for 60 minutes.

1h 15m

Transfer onto nitrocellulose in 1X NuPAGE transfer buffer with 10% methanol at 30V for 2 hours at RT. 

2h 15m

Scan nitrocellulose membrane on Licor infrared scanner with 169 µm resolution. 

Expected result
Representative image for hnRNPA1 at three different RNaseI concentrations.
 
The image shows a representative example of the membrane scan for hnRNPA1 (indicated by a purple triangle), testing three different concentrations for RNaseI (0.25, 0.025 and 0.005U). The image is shown in greyscale (left) and pseudo colouring with higher contrast (right) to show different signal intensities. Purple rectangles indicate the region processed for library preparation. 

15m

Place nitrocellulose membrane on filter paper soaked in PBS to cut out region of interest and place nitrocellulose pieces in 1.5 ml LoBind Tubes.

Note
Region of interest usually corresponds to ~20-60 kDa above the molecular weight of the RBP of interest due to the ligation of TLC-L3 adapter (~15.9 kDa) and additional weight depending on the length of associated RNA molecule (70 nt of RNA are on average 20kDa). 

15m

Step case

Omission of PAGE purification
53 steps

When omitting PAGE purification, magnetic beads bound to RNA-protein complexes in step 6.2 can be directly resuspended in 100 µl Proteinase K buffer containing 100 µg of Proteinase K and incubated at 50°C at 800 rpm for 45 minutes (see Step 7.1).
Attract beads and transfer supernatant to fresh PCR tubes containing oligo(dT) beads (see Step 7.4). 

RNA Purification

1h 30m

RNA Purification

Upon capture on oligo(dT) beads (Step 7.5), resuspend beads throughout all steps by vortexing unless otherwise stated. Depending on the reaction, oligo(dT) beads can be sticky and mixing by pipetting can cause unnecessary loss of material through retention in pipette tips. 

Add 200 µl of Proteinase K buffer containing 100 µg of Proteinase K to LoBind tube containing nitrocellulose pieces and incubate at 50°C at 800 rpm for 45 minutes.
Shaker800 rpm, 50°C, 00:45:00  

Note
1 µl of Proteinase K reaction can be dot blotted on nitrocellulose to visualise RNA release. 

Meanwhile, prepare 10 µl of oligo(dT) beads per sample and wash once in 1 ml oligo(dT) Binding Buffer.

10m

Resuspend oligo(dT) beads in 50 µl Binding Buffer per sample and distribute in fresh PCR tubes.

Transfer Proteinase K reaction to oligo(dT) beads and incubate at RT for 10 minutes rotating.

15m

Wash twice in 125 µl oligo(dT) Wash Buffer and once in 20 µl 1X First-Strand (FS) Buffer by vortexing. 

15m

Reverse Transcription

50m

Reverse Transcription and RNA elution

Prepare RT reaction and add 10 µl to beads - vortex to mix. 

Reverse Transcriptionx1x #
5X FS Buffer2 µl
10mM dNTPs0.5 µl
0.1M DTT0.1 µl
SuperaseIN0.3 µl
Superscript IV0.1 µl
H2O7 µl

10m

Incubate at 50°C for 15 minutes with interval mixing then heat up to 96°C on thermomixer. 

Note
Seal tubes with PCR plates seal to avoid lids from opening at higher temperatures on heatblock. 

25m

When 96°C is reached, vortex for 30 seconds on heatblock, spin down, and immediately place on magnet on ice.

Remove supernatant and wash beads once in 60 µl oligo(dT) Wash buffer and once in 20 µl 1X T4 RNA Ligase buffer - vortex to mix. 

Note
Supernatant can be kept to visualise the elution of RNA-TLC-L3 hybrid through dot blotting on nitrocellulose.

Expected result
Example of dot blots at different steps during the protocol to visualise the amount of adapter-ligated RNA. 

10m

Second adapter ligation

50m

Second adapter ligation

Add 5 µl of 5' adapter mix to beads and vortex to mix. 

5' Adapter Mixx1x #
10X T4 RNA Ligase Buffer2 µl
10 µM L## oligo2 µl
DMSO1 µl

Note
Ensure balanced nucleotide composition of in-read barcodes that are used at this step, by using at least four different adapters that, when multiplexed, have a balanced 'per base sequence content' for the barcode sequence.

Incubate at 75°C for 2 minutes then immediately place on ice. 

Add 4 µl of Ligation mix at the top of the tube, spin down and vortex to mix. 

Ligation mixx1x #
0.1 M ATP0.5 µl
TdT (14U/µl)0.5 µl
T4 RNA Ligase High Conc. (30U/µl)0.5 µl
H202.5 µl

Note
Beads can be sticky, avoid touching with pipette tip when adding ligation mix!

Add 10 µl PEG8000, spin down and vortex, then resuspend beads by pipetting up and down 10X at slow speed. 

10m

Incubate at 37°C for 20 minutes, then cool down to 20°C. 
Temperature37 °C   Duration00:20:00   

20m

Add 1 µl of T4 RNA Ligase (High Concentration) and pipette to mix before incubating overnight at 20°C. 
DurationOvernight   Temperature20 °C   

cDNA pre-amplification

cDNA pre-amplification

Add 100 µl of oligo(dT) Wash buffer to ligation reaction and place on magnet. 
Note
Beads don't resuspend properly at this point - apply magnetic field from different sides of the tube until beads move swiftly from one side to the other in order to aid resuspension. 

Discard supernatant and wash once more with 100 µl oligo(dT) supernatant and once in 20 µl 1X Phusion HF Buffer - vortex to mix. 

10m

Prepare cDNA pre-amplification mix and resuspend beads in 25 µl - vortex to mix. 

cDNA amplificationx1x #
2X Phusion HF PCR Mastermix12.5 µl
10 µM P7&P5 short1.25 µl
H2O11.25 µl

Amplify with the following programme: 
1. 98°C - 30 seconds
        2. 98°C - 10 seconds
        3. 65°C - 30 seconds
        4. 72°C - 30 seconds
            Go to Step #2 6 times
5. 72°C - 3 minutes
6. 16°C - HOLD

Note
When preparing libraries after omission of PAGE purification a total of 6 pre-amplification cycles are sufficient. 

20m

Wash 1-2 µl of oligo(dT) beads per sample in oligo(dT) Binding buffer and resuspend in 5 µl oligo(dT) binding buffer per sample. 

Add 5 µl of oligo(dT) beads to PCR reaction and rotate at RT for 5 minutes. 

10m

Place on magnet and transfer amplified cDNA in supernatant to a fresh tube.

Note
Pre-amplified cDNA can be stored at 4°C for short-term or -20°C for longer term.

cDNA size selection

50m

cDNA size selection

Perform ProNex Size Selection using 2.8X ProNex beads - add 84 µl of ProNEX beads and mix by pipetting up and down 10 times.

Note
The exact ratio of ProNEX beads might have to be optimised for different batches of beads. 

Incubate for 10 minutes at RT then place on magnet for 5 minutes. 

15m

Discard supernatant and wash twice in 200 µl Wash Buffer with beads remaining on the magnet. 

Remove Wash buffer and air-dry the beads on magnet until pellet starts to show cracks. 

10m

Resuspend in 23 µl H2O and incubate for 5 minutes at RT. 

10m

Place on magnet and transfer 20 µl of supernatant to a new tube. 

Expected result
Pre-amplified cDNA can be run on High Sensitivity Bioanalyser Chip as anadditional quality control step, but will most likely only show signal for strong RBPs.

PCR amplification

1h 45m

PCR amplification
Estimate the necessary number of PCR cycles for ideal library amplification by running a test qPCR on 1 µl of pre-amplified cDNA and subtract 3-4 cycles from the obtained Ct value for the final library amplification. Overamplification of TLC-CLIP libraries should be avoided to minimise the number of PCR duplicates and increase the final yield of usable reads. 

Prepare qPCR Mastermix and add 9 µl to 1 µl of cDNA. 

Test qPCRx 1x #
2X PowerUP SYBR Green Mastermix5 µl
10 µM P5 + P7 primers0.5 µl
H2O3.5 µl
Determine optimal cycle number by running the following programme:
1. 98°C - 30 seconds
            20 cycles of:
        2. 98°C - 10 seconds
        3. 68°C - 30 seconds
        4. 72°C - 30 seconds


Note
Calculate required PCR cycles for amplification by removing 3-4 cycles from the determined Ct value. 

Expected result

Amplification curves for duplicate libraries from different starting material. 

Mix 10 µl of cDNA with 30 µl of PCR Mastermix and amplify with the following programme: 
 
PCRx 1x #
2X Phusion HF Mastermix20 µl
10 µM P5 + P7 primers1 µl
H2O9 µl

1. 98°C - 30 seconds
            # of cycles:
        2. 98°C - 10 seconds
        3. 68°C - 30 seconds
        4. 72°C - 30 seconds
5. 72°C - 3 minutes
6. 16°C - HOLD


Note
Add different i7 indexes at this point to allow greater multiplexing. 

Note
10 µl of cDNA is used for PCR amplification to allow repetition in case of substantial over-amplification or unexpected size profile. 

45m

Size-selection of libraries

50m

Size-selection of libraries

Perform ProNex Size Selection using 1.8X ProNex beads - add 72 µl of ProNEX beads and mix by pipetting up and down 10 times.

Incubate for 10 minutes at RT then place on magnet for 5 minutes. 

15m

Discard supernatant and wash twice in 200 µl Wash Buffer while on magnet. 

Air-dry the beads on magnet until pellet starts to show cracks

10m

Resuspend in 17.5 µl H2O and incubate for 5 minutes at RT. 

10m

Place on magnet and transfer 15 µl of supernatant to a new tube. 

Quality Control and Quantification

Quality Control and Quantification

Prepare 1:3 or 1:5 dilution for QC and quantification.

Run 1 µl on Agilent High Sensitivity DNA Chip.

Expected result

Quantify libraries using Qubit High Sensitivity dsDNA kit. 

20m

Multiplexing and sequencing

Multiplexing and sequencing

Multiplex samples in the final pool at the desired ratios and sequence on Illumina NextSeq500 using the High Output Kit for 75 cycles with the addition of 5% PhiX. 

Demultiplexing and Trimming

Demultiplexing and Trimming of Reads using Flexbar

This is done in a two-step approach for the following reason:
The easiest way to trim 3' adapter contamination is to specifcy the entire adapter sequence (see Flexbar_2 below) with 'N' being used for the i7 index positions so that this step does not have to be run individually for different i7 indeces.

However, if this is specified together with --umi-tags, any bases on the 3' end that might correspond to the i7 index will be added to the read header and appended to the UMI which interferes with umi-tools as some UMIs will have more characters than others.

As such it is easiest to handle the demultiplexing based on In-read barcodes as well as the UMIs in the first step, and then trim remaining homopolymers at the 5' end as well as adapter contamination at the 3' end in a second step. 

Demultiplexing based on In-read barcodes

Flexbar.v3.4 was used. 
This command demultiplexes based on in-read barcodes and moves UMIs in the read header. 

-r sample.fastq.gz                # Fastq file demultiplexed based on i7 index but not trimmed
-b TLC_barcodes.fasta        # Fasta file specifying in-read barcodes and UMIs
--barcode-unassigned        # Generates file containing all unassigned barcodes
--barcode-trim-end LTAIL   # Defines barcode position within the read
--barcode-error-rate 0        # Determines number of mismatches and indels allowed in barcode
--umi-tags                            # Wildcard character 'N' specified within barcode fasta file will be appended to read name separated by underscode
-n 4                                       # Number of threads
-t path/sample_demult        # Prefix of output files
-z GZ                                    # Output files are compressed using gzip
Command
Flexbar_1
flexbar -r sample.fastq.gz -b TLC_barcodes.fasta --barcode-unassigned --barcode-trim-end LTAIL --barcode-error-rate 0 --umi-tags -n 4 -t path/sample_demult -z GZ


Example of TLC_barcodes.fasta file
Command
Fasta file containing in-read barcodes for demultiplexing with Flexbar.
>TLC_L01
NNNNNCGTGATNNNN
>TLC_L02
NNNNNACATCGNNNN
>TLC_L03
NNNNNGCCTAANNNN
>TLC_L04
NNNNNTGGTCANNNN
>TLC_L05
NNNNNCACTGTNNNN
>TLC_L06
NNNNNATTGGCNNNN
>TLC_L07
NNNNNGATCTGNNNN
>TLC_L08
NNNNNTCAAGTNNNN
>TLC_L09
NNNNNCTGATCNNNN
>TLC_L10
NNNNNAAGCTANNNN
>TLC_L11
NNNNNGCTCATNNNN
>TLC_L12
NNNNNTACAAGNNNN

Trimming of adapter and homopolymers

flexbar.v3.4 was used
This command trims remaining homopolymers at the 5' end of reads as well as adapter contamination at the 3' end. 

-r sample_demult.fastq.gz    # Demultiplexed fastq files from previous trimming step
--adapter-seq 'AGATCGGAAGAGCACACGTCTGAACTCCAGTCACNNNNNNATCTCGTATGCCGTCTTCTGCTTG'
                                                # Seqeunce of the Illumina Multiplexing Index Read Sequencing Primer with 'N' designating the position of different i7 indices.
--adapter-trim-end RIGHT   # Defines which end of the read to trim the adapter sequence off
--adapter-error-rate 0.1        # Determines how many mismatches and indels are allowed for the adapter sequence to be removed
--adapter-min-overlap 1       # Minimum required overlap for adapter to be removed
--min-read-length 18            # Discards reads shorter than 18 nucleotides after trimming
-n 2                                         # Number of threads       
--htrim-left T                         # Trims poly(T) from the left side of the read
--htrim-max-length 2           # Defines maximum length of poly(T) stretch to be trimmed as 2
--htrim-min-length 1             # Defines minimum length of poly(T) stretch as 1
-t path/sample_tr1-2             # Prefix of output files
-z GZ                                      # Output files are compressed using gzip


Command
Flexbar_2
flexbar -r sample_demult.fastq.gz --adapter-seq 'AGATCGGAAGAGCACACGTCTGAACTCCAGTCACNNNNNNATCTCGTATGCCGTCTTCTGCTTG' --adapter-trim-end RIGHT --adapter-error-rate 0.1 --adapter-min-overlap 1 --min-read-length 18 --length-dist -n 2 --htrim-left T --htrim-max-length 2 --htrim-min-length 1 -t path/sample_tr1-2 -z GZ

Remove space from read header before mapping to keep UMI
Command
zcat $sample_tr1-2.fastq.gz | tr ' ' ':' | gzip -c > path/mapready/sample_tr1-2.fastq.gz

Mapping and Deduplication

Mapping is performed with STAR, keeping only uniquely mapping reads and removing the penalty for opening deletions or insertions.

STAR mapping
STAR v.2.7.3a was used

--runThreadN 8                                    # Number of threads
-genomeDir /genome_index/STAR/    # Path to genome directory
--readFilesIn sample_tr1-2.fastq.gz    # Path to fastq file
--readFilesCommand zcat                   # Uncompession Command for compressed fastq
--outSAMtype BAM SortedByCoordinate # Output sorted by coordinate
--limitBAMsortRAM 20000000000     # Maximum available RAM for sorting BAM
--outSAMattributes All                         # Include all attributes
--outSAMunmapped Within                 # Output unmapped reads within the main SAM file
--runRNGseed 42                                 # random number generator seed
--outTmpDir {localdir}                          # path to temporary directory
--outFilterMultimapNmax 1                  # maximum number of multiple alignments allowed for a read
--outSJfilterReads Unique                    # Only uniquely mapping reads are considered for splice junction output
--alignEndsType Extend5pOfRead1    # Fully extend only the 5' end of the read, local alignment for 3' end
--scoreDelOpen 0                                 # Remove deletion open penalty
--scoreInsOpen 0                                 # Remove insertion open penalty
--outFileNamePrefix path/sample       # Prefix for output files
Command
STAR_mapping
STAR --runThreadN 8 -genomeDir /genome_index/STAR/ --readFilesIn sample_tr1-2.fastq.gz  --readFilesCommand zcat --outSAMtype BAM SortedByCoordinate --limitBAMsortRAM 20000000000 --outSAMattributes All --outSAMunmapped Within --runRNGseed 42 --outTmpDir {localdir} --outFilterMultimapNmax 1 --outSJfilterReads Unique --alignEndsType Extend5pOfRead1 --scoreDelOpen 0 --scoreInsOpen 0 --outFileNamePrefix mapping/star/sample

Deduplication with umi-tools
UMI-tools v.1.0.1 was used in dedup mode:

-I mapping/star/sample_tr1-2.bam             # mapped bam file to read
-L logs/umi_tools/umi_tools_sample.log    # file with logging information
-S mapping/star_dd/sample_dd.bam        # output file 
--extract-umi-method read_id                   # Barcodes are contained at the end of the read header
--method unique                                         # Reads group share the exact same UMI
--spliced-is-unique                                     # Two reads that start in the same position on the same strand and having the same UMI are considered unique if one is spliced and the other is not.

Command
umitools_dedup
umi_tools dedup -I mapping/star/sample_tr1-2.bam -L logs/umi_tools/umi_tools_sample.log -S mapping/star_dd/sample_dd.bam --extract-umi-method read_id --method unique --spliced-is-unique

Name	Sequence	Scale	Purification	Index
3' Adapter
TLC-L3	/5Phos/AGATCGGAAGAGCACACGTCTGAAAAAAAAAAAAAAAAAAAAAAAAA/3IR800CWN/	250nm	RNASE
5' Adapter
TLC_L01	/5Phos/NNNNATCACGNNNNNAGATCGGAAGAGCGTCGTG/3ddC/	100nm	STD	CGTGAT
TLC_L02	/5Phos/NNNNCGATGTNNNNNAGATCGGAAGAGCGTCGTG/3ddC/	100nm	STD	ACATCG
TLC_L03	/5Phos/NNNNTTAGGCNNNNNAGATCGGAAGAGCGTCGTG/3ddC/	100nm	STD	GCCTAA
TLC_L04	/5Phos/NNNNTGACCANNNNNAGATCGGAAGAGCGTCGTG/3ddC/	100nm	STD	TGGTCA
TLC_L05	/5Phos/NNNNACAGTGNNNNNAGATCGGAAGAGCGTCGTG/3ddC/	100nm	STD	CACTGT
TLC_L06	/5Phos/NNNNGCCAATNNNNNAGATCGGAAGAGCGTCGTG/3ddC/	100nm	STD	ATTGGC
TLC_L07	/5Phos/NNNNCAGATCNNNNNAGATCGGAAGAGCGTCGTG/3ddC/	100nm	STD	GATCTG
TLC_L08	/5Phos/NNNNACTTGANNNNNAGATCGGAAGAGCGTCGTG/3ddC/	100nm	STD	TCAAGT
TLC_L09	/5Phos/NNNNGATCAGNNNNNAGATCGGAAGAGCGTCGTG/3ddC/	100nm	STD	CTGATC
TLC_L10	/5Phos/NNNNTAGCTTNNNNNAGATCGGAAGAGCGTCGTG/3ddC/	100nm	STD	AAGCTA
TLC_L11	/5Phos/NNNNATGAGCNNNNNAGATCGGAAGAGCGTCGTG/3ddC/	100nm	STD	GCTCAT
TLC_L12	/5Phos/NNNNCTTGTANNNNNAGATCGGAAGAGCGTCGTG/3ddC/	100nm	STD	TACAAG
cDNA Amplification
P5short_TLC-CLIP	ACACGACGCTCTTCCGATCT	100nm	PAGE
P7short_TLC-CLIP	TGACGTGTGCTCTTCCGATCT	100nm	PAGE
PCR amplification
P5_Universal_adapter	AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT	1um	PAGE
P7-1_TLC-CLIP	CAAGCAGAAGACGGCATACGAGATCGTGATGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC*T	250nm	PAGE	ATCACG
P7-2_TLC-CLIP	CAAGCAGAAGACGGCATACGAGATACATCGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC*T	250nm	PAGE	CGATGTAT
P7-3_TLC-CLIP	CAAGCAGAAGACGGCATACGAGATGCCTAAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC*T	250nm	PAGE	TTAGGCAT
P7-4_TLC-CLIP	CAAGCAGAAGACGGCATACGAGATTGGTCAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC*T	250nm	PAGE	TGACCAAT
P7-5_TLC-CLIP	CAAGCAGAAGACGGCATACGAGATCACTGTGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC*T	250nm	PAGE	ACAGTGAT
P7-6_TLC-CLIP	CAAGCAGAAGACGGCATACGAGATATTGGCGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC*T	250nm	PAGE	GCCAATAT
P7-7_TLC-CLIP	CAAGCAGAAGACGGCATACGAGATGATCTGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC*T	250nm	PAGE	CAGATCAT
P7-8_TLC-CLIP	CAAGCAGAAGACGGCATACGAGATTCAAGTGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC*T	250nm	PAGE	ACTTGAAT

Final Concentration	Stock Solution	For 500 ml
50 mM Tris-HCl, pH 7.4	1 M Tris-HCl, pH 7.5	25 ml
100 mM NaCl	5 M NaCl	10 ml
1% Igepal-CA-630	100% Igepal CA-630	5 ml
0.1% SDS	10% SDS	5 ml
0.5% sodium deoxycholate		2.5 g
Add H2O		455 ml

Final Concentration	Stock Solution	For 10 ml
350 mM Tris-HCl, pH 6.5	1 M Tris-HCl, pH 6.5	3.5 ml
50 mM MgCl2	2 M MgCl2	0.25 ml
5 mM DTT	1M DTT	0.05 ml
Add H2O		6.2 ml

Final Concentration	Stock Solution	For 50 ml
100 mM Tris-HCl, pH 7.4	1 M Tris-HCl, pH 7.4	5 ml
50 mM LiCl	8 M LiCl	0.3125 ml
1 mM EDTA	0.5 M EDTA	0.1 ml
0.2% LiDS	1% LiDS	10 ml
Add H2O		34.5875 ml

PNK Reaction	x 1	x #
5X PNK Buffer	4 µl
SuperaseIN	0.5 µl
T4 PNK	0.5 µl
H2O	15 µl

Ligation Mix	x 1	x #
4X Ligation Buffer	5 µl
T4 RNA Ligase	1 µl
1 µM L3 Adapter	1 µl
SuperaseIN	0.5 µl
H2O	8.5 µl
PEG400	4 µl

Reverse Transcription	x1	x #
5X FS Buffer	2 µl
10mM dNTPs	0.5 µl
0.1M DTT	0.1 µl
SuperaseIN	0.3 µl
Superscript IV	0.1 µl
H2O	7 µl

5' Adapter Mix	x1	x #
10X T4 RNA Ligase Buffer	2 µl
10 µM L## oligo	2 µl
DMSO	1 µl

Ligation mix	x1	x #
0.1 M ATP	0.5 µl
TdT (14U/µl)	0.5 µl
T4 RNA Ligase High Conc. (30U/µl)	0.5 µl
H20	2.5 µl

cDNA amplification	x1	x #
2X Phusion HF PCR Mastermix	12.5 µl
10 µM P7&P5 short	1.25 µl
H2O	11.25 µl

Test qPCR	x 1	x #
2X PowerUP SYBR Green Mastermix	5 µl
10 µM P5 + P7 primers	0.5 µl
H2O	3.5 µl

PCR	x 1	x #
2X Phusion HF Mastermix	20 µl
10 µM P5 + P7 primers	1 µl
H2O	9 µl

Public workspaceTLC-CLIP

Omission of PAGE purification53 steps

TLC-CLIP

Omission of PAGE purification
53 steps