Nov 20, 2024

Public workspaceTIREseq - Turbocapture Integrated RNA Expression Sequencing

DOI
dx.doi.org/10.17504/protocols.io.j8nlk8rqdl5r/v1
  • 1Walter and Eliza Hall Institute
Daniel V Brown
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DOI: dx.doi.org/10.17504/protocols.io.j8nlk8rqdl5r/v1
Protocol Citation: Daniel V Brown 2024. TIREseq - Turbocapture Integrated RNA Expression Sequencing. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlk8rqdl5r/v1
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 19, 2024
Last Modified: November 20, 2024
Protocol Integer ID: 105897
Keywords: RNA-seq, Extraction, Transcriptomics
Funders Acknowledgement:
WEHI NMAT theme
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Abstract
To streamline sample processing we developed a novel protocol integrating RNA purification with library preparation, Turbocapture Integrated RNA Expression Sequencing (TIREseq) This new protocol exhibits a significantly accelerated workflow, higher sequencing efficiency and lower molecular contamination.
Image Attribution
Images created with Biorender.com
Guidelines
  • All reagents and plastic-ware can be found in the 'Materials' section.
  • Use only RNase free supplies and clean all surfaces and tools with RNase Away prior to working
  • Make sure all steps involving cell lysate and RNA before reverse transcription are carried out swiftly and on ice.
  • Universal primer sequences are listed below:

ABC
NameSequenceNotes
TIRE_TSO /5Biosg/AGAGACAGATTGCGCAATGNNNNNNNNNNWWrGrGrG5' biotin. 3x 3' RNA 'G'. Order HPLC purified
TIRE hexamerCTACACGACGCTCTTCCGATCTGANNNGGNN*N*BDesalted, 3' phosphorothioate bonds
TIRE PCR1 FwdAGTTCAGACGTGTGCTCTTCCGAT*C*TDesalted, 3' phosphorothioate bonds
TIRE PCR1 RevCTACACGACGCTCTTCCGAT*C*TDesalted, 3' phosphorothioate bonds
Universal primer sequences

Well barcoding primer sequences: Download TIRE_read1_wellBC.csvTIRE_read1_wellBC.csv
SI-TT plate primer sequences: Download Dual_Index_Kit_TT_Set_A.csvDual_Index_Kit_TT_Set_A.csv
Combination of plate and well barcoding sequences: Download plate_and_well_BCs.csvplate_and_well_BCs.csv

Materials
ReagentSPRIselect reagent kitBeckman CoulterCatalog #B23317
Reagentbeta mercaptoethanolContributed by users
ReagentHotStart ReadyMix (KAPA HiFi PCR kit)Kapa BiosystemsCatalog #KK2601
ReagentMaxima H Minus Reverse TranscriptaseThermo Fisher ScientificCatalog ##EP0741
ReagentDNA Polymerase I Klenow Fragment - 1,000 unitsNew England BiolabsCatalog #M0210L
Reagent10 mM dNTPsLife TechnologiesCatalog #10297-018
ReagentSUPERase• In™ RNase Inhibitor (20 U/μL)Thermo Fisher ScientificCatalog #cat# AM2694
ReagentBuffer TCL QiagenCatalog #1031576
ReagentBuffer TCL, 2x QiagenCatalog #1070498
ReagentTurboCapture 96 mRNA KitQiagenCatalog #72251
ReagentUltrapure Distilled, Nuclease Free WaterContributed by users
Reagent0.2 ml PCR Tube stripsEppendorfCatalog #0030124359
ReagentNalgene™ Disposable Polypropylene Robotic Reservoirs, flatThermo FisherCatalog #1200-1300
ReagentAdhesive PCR Plate SealsThermo Fisher ScientificCatalog #AB0558

Safety warnings
Please follow all Manufacturer safety warnings and recommendations.
Keep a separation of pre and post PCR steps in separate work areas.
Before start
Wipe bench surfaces with PCR clean wipes and keep working environment clean.
Cell lysis
Cell lysis
30m
30m
Prepare TCL lysis buffer

Note
The most concentrated lysate I have made is 2,000 cells per uL. Beyond this concentration the lysate becomes too viscous from genomic DNA.

Toxic
For adherent cells prepare enough 1x TCL to cover the surface of the culture dish. Add 1% v/v 2-Mercaptoethanol.

For suspension cells prepare an equal volume of 2x TCL as the cell culture media volume. Add 1% v/v 2-Mercaptoethanol.
Prepare cell lysate
For adherent cells, remove cell culture media by vacuum and add 1 volume of PBS to wash.
After removing PBS, add the relevant volume of 1x TCL buffer based on estimate cell number.
Use a cell scraper and p1000 pipette to homogenize lysate.
For suspension cells, add the relevant volume of 2x TCL buffer directly to culture media.
Use a p1000 pipette to homogenize lysate.
The lysate may be stored for at least 6 months at Temperature-80 °C without any loss in performance.

TIRE-seq preparation
TIRE-seq preparation
5m
5m
Clean all surfaces and pipettes with PCR clean wipes
Turn on a lab oven to Temperature50 °C and another to Temperature80 °C
Thaw frozen buffers and primers on ice
Prepare fresh 80% EtOH
mRNA capture
mRNA capture
2h 45m
2h 45m
Thaw the cell lysates TemperatureOn ice

1m
Transfer Amount20 µL cell lysate to a TurboCapture plate.
I use a 96w pipetting robot for this step.

Note
Avoid creating bubbles or foam.
A maximum of 80uL cell lysate may be used but be aware an equal volume of Kappa HiFi polymerase should be added at a later step driving up costs.

10m
Cover the plate with a plastic seal and incubate at TemperatureRoom temperature for Duration01:00:00 on a laboratory rocker

1h
Remove the liquid from the TurboCapture plate by flicking over a sink. Use the ELISA flicking technique.
Dilute out the liquid in the sink by running the tap.
1m
Wash the TurboCapture plate using 100 μl Buffer TCW per well.
Flick the TCW into the sink
Note
Pipetting accuracy not critical here.
Prepare the reverse transcriptase mix before the final wash so the TurboCapture plate does not dry out.

5m
Wash
Go togo to step #12 repeat the wash an additional 2 more times for a total of 3 washes

5m
Reverse transcription
Reverse transcription
2h 45m
2h 45m
Prepare reverse transcriptase mixture:

ABCD
Reagent1 rxn96 rxn + 20%Conc
5x Maxima buffer4460.81x
100mM dNTPs0.2231mM
100uM TIRE_TSO0.2231uM
20U/uL SuperaseIN0.2230.2U/uL
200U/uL Maxima reverse transcriptase2230.4100U
Water13.41543.7
Total202304

5m
Remove all buffer TCW from the TurboCapture plate by pulse centrifugation and aspirating with a multi channel pipette.
10m
Critical
Add Amount20 µL reverse transcription mix
Incubate in a thermocycler for:
  1. Temperature50 °C 1 hour
  2. Temperature80 °C 10min
  3. Temperature4 °C Hold

1h 20m
cDNA denaturing
cDNA denaturing
25m
25m
Add Amount150 µL Concentration100 millimolar (mM) NaOH to the plate with reverse transcription mixture and immediately flick out the liquid over the sink.

5m
Add Amount100 µL
Concentration100 millimolar (mM) NaOH to the plate. Incubate for Duration00:05:00 room temp


5m
Wash with Amount150 µL Concentration10 millimolar (mM) Tris pH 7.5
Flick out the liquid over the sink.
5m
Go togo to step #19
Repeat wash for a total of 2 washes
Note
Have Kappa HiFi and the well barcoding primers thawed and ready so the plate does not dry out

5m
Remove all Tris buffer from the TurboCapture plate by pulse centrifugation and aspirating with a multi channel pipette.
5m
Critical
Linear amplification
Linear amplification
1h 20m
1h 20m
Dispense Amount10 µL 2x Kappa HiFi Polymerase into each well with a repeat dispenser or multichannel pipet.

5m
Dispense Amount10 µL of Concentration1 micromolar (µM) barcoding primer to each well. Mix well.
I use a 96w pipetting robot for this step.

10m
Incubate in a thermocycler:


ABCD
StepTemperatureTimeCycles
Inital denaturation982min1
Denature9820sec10 cycles
Anneal6030sec
Extension7230sec
Final elongation722min1
Hold
Linear amplification protocol. Sample can be held overnight.


1h
Pause
Sample pooling and purification
Sample pooling and purification
1h 20m
1h 20m
Prepare SPRI beads by diluting SPRIselect commerical beads 1:4 in SPRI buffer.
Prepare Amount2.5 mL diluted SPRI beads per 96 well plate.

Pool all wells together. You can invert the plate into a SBS reservoir and centrifuge.
Or use a multichannel pipette.
After pooling, mix the sample well in a single tube then distribute across 4x 1.5mL eppendorf tubes.
Measure the volume of each tube with a p1000 pipette. Use the dialing down method.
Perform a 1.2x SPRI bead : sample cleanup.
For example for 450uL of sample add Amount540 µL diluted SPRI beads.

Incubate for Duration00:05:00 at room temp

5m
Incubate on a magnet for Duration00:05:00 at room temp

5m
Keep tubes on magent and remove the supernatant carefully.
Add Amount500 µL 80% ethanol

Go togo to step #31 for a total of 2 ethanol washes.

After removing supernatant perform a pulse centrifuge and remove all residual ethanol with a pipet
Incubate on the magnet for Duration00:02:00

2m
Elute the sample with Amount30 µL water
Transfer Amount28 µL supernatant of each of the 4 aliquots into separate positions of a 0.2mL strip tube.
Sample can be stored for several weeks at Temperature-20 °C

Pause
Library preparation
Library preparation
1h
1h
Keep half the sample at Temperature-20 °C as a backup.
Prepare the following random hexamer master mix:

ABC
Reagent1 rxn4 rxn + 10%
10x NEB buffer 3.128.8
100uM TIRE hexamer primer28.8
10mM dNTPs28.8
Linear amp product13
Total1983.6


Add random hexamer mix to the samples and incubate Temperature95 °C for 2 min in a thermocycler.

Remove from thermocycler and allow the sample to cool to room temperature.
Add Amount1 µL Large (Klenow) Fragment and mix well.

Incubate at Temperature25 °C for 30min

30m
Incubate at Temperature75 °C for 20min

20m
Perform a 1.8x SPRI bead cleanup with undiluted beads with the sample protocol as Go togo to step #29

Elute in Amount20 µL water in a 0.2mL strip tube.

First PCR amplification
First PCR amplification
2h
2h
Prepare a PCR master mix for each of the 4 aliquots from the Klenow reaction:

ABCD
Reagent1 rxn4 rxn + 10%Conc
Purified Klenow reaction20
2x Kappa HiFi polymerase25110
10uM each TIRE PCR1 primer premix522400nM
Total50220
PCR1 master mix

Add Amount30 µL PCR1 master mix to each purified Klenow reaction.

Run the following PCR protocol:
ABCD
StepTemperatureTimeCycles
Inital denaturation982min1
Denature9820sec10 cycles*
Anneal6030sec
Extension7230sec
Final elongation722min1
Hold
PCR1 cycling conditions
Note
*I have performed experiments comparing PCR cycle numbers in the PCR1 and PCR2 steps.
10 PCR cycles has worked well for 1 million - 10 million cells across the 96w plate
For 100,000 cells total I use 12 PCR cycles.
I found it better to err on the side of more PCR cycles in the 1st PCR step and less in the 2nd PCR step.
Pause
Perform a 1x SPRI bead cleanup with undiluted beads following the protocol in Go togo to step #29 PCR cleanup

Elute in Amount15 µL EB and transfer Amount13 µL to a new tube.

Second PCR amplification
Second PCR amplification
5m
5m
Keep the 4 tubes separate for this step but use the same index primer set.
2h
Critical
Prepare a PCR master mix for each of the 4 aliquots from the PCR 1 reaction:
ABC
Reagent1 rxn4 rxn + 10%
Purified PCR1 reaction5
2x Kappa HiFi polymerase12.555
Index set SI-TT-xx522
water2.511
Total25220
PCR2 master mix
Keep Run the following PCR protocol:
ABCD
StepTemperatureTimeCycles
Inital denaturation982min1
Denature9820sec8 cycles*
Anneal5430sec
Extension7220sec
Final elongation721min1
Hold
PCR2 cycling conditions
Note
*I have performed experiments comparing PCR cycle numbers in the PCR1 and PCR2 steps.
8 PCR cycles has worked well for 1 million - 10 million cells across the 96w plate
For 100,000 cells total I use 10 PCR cycles.
I found it better to err on the side of more PCR cycles in the 1st PCR step and less in the 2nd PCR step.

Pause
Perform a 2 sided SPRI bead size selection to obtain compatible fragments for Illumina sequencing.
Combine all 4 aliquots into a single tube ~ Amount100 µL Crude PCR2 product

Add Amount50 µL (0.5x) undiluted SPRI beads to the pooled sample.

Incubate for Duration00:05:00 at room temp

5m
Incubate on a magnet for Duration00:05:00 at room temp

5m
Keep the supernatant! Transfer cleared supernatant to a new 1.5mL tube.
Critical
Add Amount25 µL (0.8x) undiluted SPRI beads and incubate off magnet for Duration00:05:00

5m
Transfer to a magnet and incubate Duration00:05:00

5m
Remove and discard supernatant. Add Amount500 µL 80% ethanol while the sample is still on the magnet.

Repeat the ethanol wash Go togo to step #51.7 for a total of 2 washes.

Pulse centrifuge to collect the liquid to the bottom of the tube.
Incubate on magnet with the cap open for Duration00:02:00

2m
Elute in Amount27 µL EB
Transfer Amount25 µL to a new tube as a finished library.

Quality control
Quality control
15m
15m
Quantify the final library by Qubit DNA high sensitivity kit.
Expect a concentration of between 1 - 20ng/uL.

Expected result

Example tapestation trace of finished library on D1000 tape




Sequencing
Sequencing
The minimum sequencing requirement is:
  1. Read 1: 50nt
  2. Index 1: 8nt
  3. Index 2: 8nt
  4. Read 2: 72nt

Note
Read 1 is the transcript and can be increased if kit capacity allows.
Read 2 contains the well barcode, TSO sequence and UMI.
I typically begin by sequencing 2 million reads per well ~ 200M reads per 96w plate.

Bioinformatic preprocessing
Bioinformatic preprocessing
The fastqs may be preprocessed with your pipeline of choice. I prefer zUMIs where the parameters are:


Command
zUMI config.yaml
sequence_files:
  file1:
    name: path_to_read2.fastq.gz
    base_definition:
      - BC(1-10)
      - UMI(28-37)
  file2:
    name: path_to_read1.fastq.gz
    base_definition:
      - cDNA(1-50)


When I have multiple plates in a run I have bclconvert write the index reads to file and concatenate fastqs from all plates into a single file:


Command
concatenate multiplate plate fastqs
cat *I1_001.fastq.gz > combined_S1_I1_001.fastq.gz
cat *R1_001.fastq.gz > combined_S1_R1_001.fastq.gz
cat *R2_001.fastq.gz > combined_S1_R2_001.fastq.gz

I then use the index read as the first part of the well barcode:


Command
zUMI multi plate.yaml
sequence_files:
  file1:
    name: path_to_index_read1.fastq.gz
    base_definition:
      - BC(1-8)
  file2:
    name: path_to_read2.fastq.gz
    base_definition:
      - BC(1-10)
      - UMI(28-37)
  file3:
    name: path_to_read1.fastq.gz
    base_definition:
      - cDNA(1-50)



You will need to create a well barcode whitelist by concatenating the index reads used to all well barcodes:

Command
concatenate_plate_well_barcodes.R
# Read index read 1 barcodes
plateBC <- read.csv(here::here(
  "i7_only_Kit_TT_Set_A.csv"
))

# Read well barcodes
wellBC <- read.csv(here::here(
  "barcodeOrder_v1.csv"
))

# Define the vectors to be concatenated
vector1 <- plateBC$i7_8nt
vector2 <- wellBC$Well_BC

# Get all combinations
combinations <- expand.grid(vector1, vector2)

# Concatenate the combinations
combinations <- as.data.frame(paste(combinations$Var1, combinations$Var2, sep=""))

# Write the result to a CSV file
write.csv(combinations, file = here::here("all_sample_BCs.csv"), 
          row.names = FALSE, quote = F, col.names = FALSE)