Brad-seq mRNA (for Shotgun or DGE)

Yin-Tse Huang

May 12, 2022

Version 2

Brad-seq mRNA (for Shotgun or DGE) V.2

This protocol is a draft, published without a DOI.

Yin-Tse Huang¹

¹Kyoto University

Yin-Tse Huang

Kaohsiung Medical University

Protocol Citation: Yin-Tse Huang 2022. Brad-seq mRNA (for Shotgun or DGE). protocols.io https://protocols.io/view/brad-seq-mrna-for-shotgun-or-dge-bqznmx5eVersion created by Yin-Tse Huang

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: In development

We are still developing and optimizing this protocol

Created: December 23, 2020

Last Modified: September 13, 2023

Protocol Integer ID: 45838

Disclaimer

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Abstract

Brad-seq mRNA

Materials

Lysis/binding buffer (LBB) 
ABC
ComponentStockVolumes for 50 ml
100 mM Tris-HCl1 M pH 85 ml
1M LiCl8 M6.25 ml
10 mM EDTA500 mM pH 81 ml
1% SDS (or LiDS)5% w/v10 ml
5 mM DTT0.5 M500 μl
Antifoam A750 ul
RNAse-free H2OMake up to 50 ml
1. Add 5 µl/ml 2-Mercaptoethanol per ml before use.
2. Store at 4 C; warm up to RT by placing at 37 C before use (make sure salt crystals are all dissolved)
3. Shelf life: 1-2 months
 
Washing Buffer A (WBA)
ABC
ComponentStockVolumes for 50 ml
10 mM Tris-HCl1 M pH 8500 μl
150 mM LiCl8 M940 μl
1 mM EDTA500 mM pH 8100 μl
0.1% SDS5% w/v500 μl
RNAse-free H2OMake up to 50 ml
Store at 4 C and keep on ice prior to use
 
Washing Buffer B (WBB) (Store at 4 C and keep on ice prior to use)

ABC
ComponentStockVolume for 50 ml
10 mM Tris-HCl1 M pH 8500 μl
150 mM LiCl8 M940 μl
1 mM EDTA500 mM pH 8100 μl
RNAse-free H2OMake up to 50 ml
Store at 4 C and keep on ice prior to use

Low-salt Buffer (LSB) (Store at 4 C and keep on ice prior to use)

ABC
ComponentStockVolume for 50 ml
10 mM Tris-HCl1 M pH 8500 μl
150 mM NaCl5 M1.5 ml
1 mM EDTA500 mM pH 8100 μl
RNAse-free H2OMake up to 50 ml
Store at 4 C and keep on ice prior to use

10 mM Tris-HCl pH 8 (Store at room temperature)
ABC
ComponentStockVolume for 50 ml
10 mM Tris-HCl1 M pH 8500 μl
RNAse-free H2OMake up to 50 ml
Store at room temperature

1 M (1000 mM) 2-Mercaptoethanol
ABC
ComponentStockVolume for 50 ml
1 M 2-Mercaptoethanol14.3 M7 μl
RNAse-free H2O93 μl
Store at -20 °C immediately after use

Ampure XP Bead Resuspension Buffer (ABR)
ABC
ComponentStockVolume for 5 ml (aimed concentration)
PEG 80001.5 ml (15%)
NaCl2.5 ml (2.5 M)
RNAse-free H2O1 ml
Store at room temperature
Remake after a few months; Deteriorate over time, will not DNA well

Protocol materials

ReagentRNase-Free DNase SetQiagenCatalog #79254Step 22.2
 

Tissue Lysis

Add Amount5 µL   of 2-Mercaptoethanol (2-ME) to Amount1 mL   of LBB (for ratio, adjust it for the amount LBB used)

Wipe off RNAlatter from tissue; Place Amount20 mg  of tissue in crushing tube with metal cone
 

Add Amount200 µL (100/10: LBB/Tissue ratio)  in tube

Crush sample with multi-beads shocker at Centrifigation2000 rpm , 2-4 times  

Sit in TemperatureRoom temperature   for Duration00:05:00    and remove metal cone

Centrifuge at Centrifigation14000 rpm, 00:01:00   

Transfer all the lysate to a new 1.5 mL tube

Centrifuge at Centrifigation14000 rpm, 00:10:00  

Carefully transfer the supernatant to a new tube. Be careful not to carry over cell debris

Note
Stop here and store samples in Temperature-80 °C   if needed

1st mRNA extraction

10m

Put Amount100 µL   lysed sample in each well of 8-strip (Keep another half in Temperature-80 °C   just in case)

Add Amount2 µL (6.25 µm)  of biotin-20nt-20T oligo; Mix well by pipetting

Sit in TemperatureRoom temperature   for Duration00:10:00   for incubation

10m

While waiting, prepare NEB magnetic Streptavidin beads
Resuspend beads well before use

Dispense Amount20 µL   of Streptavidin beads into each well of a 8-strip (Amount1 mg  :Amount1 µL   beads/tissue ratio); Put 8-strip on magnetic rack and remove supernatant

Resuspend beads with Amount100 µL   LBB to wash the beads;
Place 8-strip on magnet rack and remove supernatant
Beads are ready for use

Add biotin-incubated samples to washed beads

Slowly stir at TemperatureRoom temperature    at Centrifigation500 rpm  for Duration00:10:00   

10m

Place 8-strip on magnet rack and remove supernatant
Note
If DNA is needed, keep the supernatant


Safety information
2-ME in the solution

Wash with Amount150 µL   of cold WBA (keep it as cool as possible) and Place 8-strip on magnet rack and remove supernatant

Wash with Amount150 µL   of cold WBB (keep it as cool as possible) and Place 8-strip on magnet rack and remove supernatant

Wash with Amount150 µL   of cold LSB (keep it as cool as possible) and Place 8-strip on magnet rack and remove supernatant

RNA elution buffer prep.
ABC
ComponentStock conc.Volume for 1 mL
Tris-HCl10 mM999 μl
2-Mercaptoethanol1 M1 μl
Freshly make every time before use

Resuspend beads in Amount17 µL   RNA elution buffer

Warm at Temperature80 °C   for Duration00:02:00    in a thermal cycler. After that, cool it quickly on ice for Duration00:05:00    
Note
Keep TTT away from AAA

Place 8-strip on magnet rack and transfer supernatant (Amount17 µL   ) to a new 8-strip
Note
Supernatant here is GOOD for DGE protocol

DNAase treatment for Secondary mRNA Recovery (for SHO protocol)

ReagentRNase-Free DNase SetQiagenCatalog #79254  
DANase prep.
AB
ComponentVolume
RDD buffer1.85 uL
DNAase I0.46 uL
total2.31 uL
DNAase (1500 K units): add 550 uL DEPC water, divided in small amount in tubes for use; shelf life 9 months

Add Amount2.31 µL   DNAase to 1st RNA supernatant (Amount17 µL   ) = Amount19.31 µL   in total

Sit at TemperatureRoom temperature   for Duration00:15:00   

15m

Kill the activity of DNAase at Temperature70 °C   for Duration00:10:00   

10m

2nd mRNA recovery

10m

Add Amount150 µL   DEPC water to re-suspend the used beads; 
Place 8-strip on magnet rack and remove supernatant

Add Amount5 µL   of 2-Mercaptoethanol (2-ME) to Amount1 mL   of SBB (for ratio, adjust it for the amount SBB used)

Add Amount150 µL   SBB and Place 8-strip on magnet rack and remove supernatant (Wash beads)

Add in DNAase treated RNA (Amount19.31 µL   ) to the washed beads + Amount130 µL   SBB

Sit at TemperatureRoom temperature   for Duration00:10:00   

10m

Place 8-strip on magnet rack and remove supernatant

Wash with Amount150 µL   of cold WBA (keep it as cool as possible) and Place 8-strip on magnet rack and remove supernatant

Wash with Amount150 µL   of cold WBB (keep it as cool as possible) and Place 8-strip on magnet rack and remove supernatant

Wash with Amount150 µL   of cold LSB (keep it as cool as possible) and Place 8-strip on magnet rack and remove supernatant

Resuspend beads in Amount17 µL   RNA elution buffer

Warm at Temperature80 °C   for Duration00:02:00    in a thermal cycler. After that, cool it quickly on ice for Duration00:05:00    
Note
Keep TTT away from AAA

Place 8-strip on magnet rack and transfer supernatant (Amount17 µL   ) to a new 8-strip
Note
Supernatant here is GOOD for SHO protocol

Note
Can be stored at Temperature-20 °C   if needed

RNA fragmentation & 3-prime adapter cDNA priming

Make 3 strand priming Amount2.5 µL   

AB
ComponentVolume
5X Thermo Scientific RT buffer1.5 μl
3-prime priming adapter1 μl
DGE 3' priming adaptor	L-3ILL-20TV.2	GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTTTTTTTTTTTTTTTTTTTV
Shotgun 3' priming adaptor	L-3ILL-N8.2	GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTNNNNNNNN

Mix Amount2.5 µL  3 strand priming with Amount7.5 µL   extracted RNA =  Amount10 µL   in total
Keep the remaining atTemperature-20 °C   )

Amount10 µL   of mixed in thermo cycler for RNA fragmentation
Note
Fragmentation/Priming program: for DGE 
(Temperature25 °C 1s  , Temperature94 °C 1.5 min  , Temperature30 °C 1 min  , Temperature20 °C 4 min  , Temperature20 °C hold  )


Note
Fragmentation/Priming program: for SHO
(Temperature25 °C 1s  , Temperature94 °C 1.5 min  , Temperature4 °C 5  min  , Temperature4 °C hold  )

Amount5 µL   master mix + Amount10 µL   fragmented RNA = Amount15 µL   mixed solution
1st strand master mix
AB
Componentvol/rxn
5X Thermo Scientific RT buffer1.5 μl
0.5M DTT0.3 μl
H2O2.2 μl
25mM dNTPs0 .5 μl
RevertAid RT enzyme0.5 μl
DTT: RNAase inhibitor
RevertAid RT enzyme add right before use

Mixed solution in thermo cycler for reverse transcription
Note
Condition:
Temperature25 °C 10min  , Temperature42 °C 50min  , Temperature50 °C 10min  , Temperature70 °C 10min  , Temperature4 °C hold  

Use Ampure beads solution for cDNA purification;
Amount35 µL   Ampure bead solution + Amount20 µL   cDNA
Ampure bead solution
AB
ComponentVolume
50 mM EDTA pH 8.05 μl
Ampure beads30 μl
Ampure bead = 1.5X sample (3:2)

Vortex for Duration00:05:00   at TemperatureRoom temperature   

Place 8-strip on magnet rack and remove supernatant

Wash with Amount200 µL   ethanol without suspending; Dry the pellet
Repeat this process twice 

Note
Don't dry the pellet too much at 2nd time, otherwise it's hard to elute


Note
Not recommend to store at Temperature-20 °C   at this stage

5-prime adapter sequence addition

15m

Add Amount4 µL   of 10 μM 5-prime adapter directly to the pellet atTemperatureRoom temperature    to resuspend the bead pellet

Prepare the master mix in advance during cDNA synthesis, and add the enzyme just before using
Amount6 µL   mater mix/rxn + Amount4 µL   suspended cDNA pellet
mater mix recipe
AB
ComponentVolume
H2O3.5 μl
10X PolI buffer1 μl
250 mM MgCl21 μl
25 mM dNTPs0 .25 μl
DNA Pol I0 .25 μl
DNA Pol I add right before use

Sit at TemperatureRoom temperature   for Duration00:15:00   

15m

Amount40 µL   Ampure bead solution + Amount10 µL   cDNA 
Ampure bead solution recipe
AB
ComponentVolume
50 mM EDTA pH 8.010 μl
Ampure Bead Resuspension Buffer (ABR)30 μl
Ampure bead = 1.5X sample (3:2)

Vortex for Duration00:05:00   at TemperatureRoom temperature   

Place 8-strip on magnet rack and remove supernatant

Wash with Amount200 µL   ethanol without suspending; Dry the pellet
Repeat this process twice 

Note
Don't dry the pellet too much at 2nd time, otherwise it's hard to elute

Elute the pellet in Amount20 µL   of 10 mM Tris pH 8.0; 
Let it sit for Duration00:01:00  

Note
Amount12 µL   when sample is little

Transfer the supernatant to new tubes

Enrichment and adapter extension

Amount12.2 µL   Enrichment master mix + Amount2 µL   1 μM ILL-INDEX primer +  Amount5.8 µL   cDNA
Enrichment master mix recipe
AB
ComponentVolume
2 X KAPA10 μl
2 μM PE1 primer1 μl
8 μM each EnrichS1 + S2 primers1 μl
25mM dNTPs0.2 μl

Mixed solution in thermo cycler

Note
Condition for SHO
Temperature98 °C 5 min  , (Temperature98 °C 20 s  , Temperature65 °C 15 s  , Temperature72 °C 15 s  ) X 18 cycles, Temperature72 °C 3 min , Temperature10 °C hold  

Condition for DGE
Temperature98 °C 5 min  , (Temperature98 °C 20 s  , Temperature65 °C 15 s  , Temperature72 °C 15 s  ) X 14 cycles, Temperature72 °C 3 min , Temperature10 °C hold  

Final Cleanup

Amount24 µL  Ampure beads + Amount20 µL  of enrichment product; 
Mix well

Vortex for Duration00:05:00   at TemperatureRoom temperature   

Place 8-strip on magnet rack and remove supernatant

Wash with Amount200 µL   ethanol without suspending; Dry the pellet
Repeat this process twice 

Note
Don't dry the pellet too much at 2nd time, otherwise it's hard to elute

Elute the pellet in Amount12 µL   of H2O

Use Bioanalyzer for quantification for measuring the concentration of each sample

Library preparation

10m

Mix equimolar of samples in a 1.5 mL tube

Add same amount of AMpure XP as the DNA, mix well, and let it stand for Duration00:05:00  

Place on magnet rack for Duration00:05:00  , remove supernatant and wash twice with Amount70 µL   80% EtOH. Dry.

Resuspend the beads by adding Amount22 µL   of water, leave them in the magnet rack for Duration00:05:00   
Transfer the supernatant to a new 0.5 mL low bind tube.

Quantification with Qbit or Bioanalyzer

A	B	C
Component	Stock	Volumes for 50 ml
100 mM Tris-HCl	1 M pH 8	5 ml
1M LiCl	8 M	6.25 ml
10 mM EDTA	500 mM pH 8	1 ml
1% SDS (or LiDS)	5% w/v	10 ml
5 mM DTT	0.5 M	500 μl
Antifoam A		750 ul
RNAse-free H2O		Make up to 50 ml

A	B	C
Component	Stock	Volume for 50 ml
10 mM Tris-HCl	1 M pH 8	500 μl
150 mM LiCl	8 M	940 μl
1 mM EDTA	500 mM pH 8	100 μl
RNAse-free H2O		Make up to 50 ml

A	B	C
Component	Stock	Volume for 5 ml (aimed concentration)
PEG 8000		1.5 ml (15%)
NaCl		2.5 ml (2.5 M)
RNAse-free H2O		1 ml

A	B	C
Component	Stock conc.	Volume for 1 mL
Tris-HCl	10 mM	999 μl
2-Mercaptoethanol	1 M	1 μl

	A	B
	Component	Volume
	RDD buffer	1.85 uL
	DNAase I	0.46 uL
	total	2.31 uL

	A	B
	Component	Volume
	5X Thermo Scientific RT buffer	1.5 μl
	3-prime priming adapter	1 μl

	A	B
	Component	vol/rxn
	5X Thermo Scientific RT buffer	1.5 μl
	0.5M DTT	0.3 μl
	H2O	2.2 μl
	25mM dNTPs	0 .5 μl
	RevertAid RT enzyme	0.5 μl

	A	B
	Component	Volume
	H2O	3.5 μl
	10X PolI buffer	1 μl
	250 mM MgCl2	1 μl
	25 mM dNTPs	0 .25 μl
	DNA Pol I	0 .25 μl

	A	B
	Component	Volume
	50 mM EDTA pH 8.0	10 μl
	Ampure Bead Resuspension Buffer (ABR)	30 μl

	A	B
	Component	Volume
	2 X KAPA	10 μl
	2 μM PE1 primer	1 μl
	8 μM each EnrichS1 + S2 primers	1 μl
	25mM dNTPs	0.2 μl

Public workspaceBrad-seq mRNA (for Shotgun or DGE) V.2

Brad-seq mRNA (for Shotgun or DGE) V.2