TITLE: Brad-seq mRNA (for Shotgun or DGE)
AUTHORS: Yin-Tse Huang

[STEPS]
?. [Tissue Lysis]
Wipe off RNAlatter from tissue; Place of tissue in crushing tube with metal cone
20 mg
?. [Tissue Lysis]
Add  of 2-Mercaptoethanol (2-ME) to  of LBB (for ratio, adjust it for the amount LBB used)
5 µl
1 mL
?. [Tissue Lysis]
Add  in tube
[100/10: LBB/Tissue ratio]
?. [Tissue Lysis]
Crush sample with multi-beads shocker at
Centrifuge: 2000 33
?. [Tissue Lysis]
Sit in  for   and remove metal cone
0 Room temperature
?. [Tissue Lysis]
Centrifuge at
Centrifuge: 14000 33, 1 min
?. [Tissue Lysis]
Transfer all the lysate to a new 1.5 mL tube
?. [Tissue Lysis]
Centrifuge at
Centrifuge: 14000 33, 10 min
?. [Tissue Lysis]
Carefully transfer the supernatant to a new tube. Be careful not to carry over cell debris
Stop here and store samples in   if needed
?. [1st mRNA extraction]
Put  lysed sample in each well of 8-strip (Keep another half in  just in case)
100 µl
-80 °C
?. [1st mRNA extraction]
Add  of biotin-20nt-20T oligo; Mix well by pipetting
6.25 µl
?. [mRNA extraction]
Sit in  for
0 Room temperature
?. [1st mRNA extraction]
While waiting, prepare NEB magnetic Streptavidin beadsResuspend beads well before use
?. [1st mRNA extraction]
Dispense  of Streptavidin beads into each well of a 8-strip (: beads/tissue ratio); Put 8-strip on magnetic rack and remove supernatant
20 µl
1 mg
1 µl
?. [1st mRNA extraction]
Resuspend beads with  LBB to wash the beads;Place 8-strip on magnet rack and remove supernatantBeads are ready for use
100 µl
?. [1st mRNA extraction]
Sit in  for  for incubation
0 Room temperature
?. [1st mRNA extraction]
Add biotin-incubated samples to washed beads
?. [1st mRNA extraction]
Slowly stir at   at  for
0 Room temperature
Centrifuge: 500 33
?. [1st mRNA extraction]
Place 8-strip on magnet rack and remove supernatant
If DNA is needed, keep the supernatant
2-ME in the solution
?. [1st mRNA extraction]
Wash with  of cold WBA (keep it as cool as possible) and Place 8-strip on magnet rack and remove supernatant
150 µl
?. [1st mRNA extraction]
Wash with  of cold WBB (keep it as cool as possible) and Place 8-strip on magnet rack and remove supernatant
150 µl
?. [1st mRNA extraction]
Wash with  of cold LSB (keep it as cool as possible) and Place 8-strip on magnet rack and remove supernatant
150 µl
?. [1st mRNA extraction]
RNA elution buffer prep.ComponentStock conc.Volume for 1 mLTris-HCl10 mM999 μl2-Mercaptoethanol1 M1 μl
ComponentStock conc.Volume for 1 mLTris-HCl10 mM999 μl2-Mercaptoethanol1 M1 μl
?. [1st mRNA extraction]
Warm at  for   in a thermal cycler. After that, cool it quickly on ice for
80 °C
Keep TTT away from AAA
?. [1st mRNA extraction]
Place 8-strip on magnet rack and transfer supernatant ( ) to a new 8-strip
17 µl
Supernatant here is GOOD for DGE protocol
?. [1st mRNA extraction]
DNAase treatment for Secondary mRNA Recovery (for SHO protocol)
?. [1st mRNA extraction]
DANase prep.ComponentVolumeRDD buffer1.85 uLDNAase I0.46 uLtotal2.31 uL
ComponentVolumeRDD buffer1.85 uLDNAase I0.46 uLtotal2.31 uL
?. [1st mRNA extraction]
Add  DNAase to 1st RNA supernatant ( ) =  in total
2.31 µl
17 µl
19.31 µl
?. [1st mRNA extraction]
Sit at  for
0 Room temperature
?. [1st mRNA extraction]
Kill the activity of DNAase at  for
70 °C
?. [2nd mRNA recovery]
Add  DEPC water to re-suspend the used beads; Place 8-strip on magnet rack and remove supernatant
150 µl
?. [2nd mRNA recovery]
Add  SBB and Place 8-strip on magnet rack and remove supernatant (Wash beads)
150 µl
?. [2nd mRNA recovery]
Add  of 2-Mercaptoethanol (2-ME) to  of SBB (for ratio, adjust it for the amount SBB used)
5 µl
1 mL
?. [2nd mRNA recovery]
Add in DNAase treated RNA ( ) to the washed beads +  SBB
19.31 µl
130 µl
?. [2nd mRNA recovery]
Sit at  for
0 Room temperature
?. [2nd mRNA recovery]
Place 8-strip on magnet rack and remove supernatant
?. [2nd mRNA recovery]
Wash with  of cold WBA (keep it as cool as possible) and Place 8-strip on magnet rack and remove supernatant
150 µl
?. [2nd mRNA recovery]
Wash with  of cold WBB (keep it as cool as possible) and Place 8-strip on magnet rack and remove supernatant
150 µl
?. [2nd mRNA recovery]
Wash with  of cold LSB (keep it as cool as possible) and Place 8-strip on magnet rack and remove supernatant
150 µl
?. [2nd mRNA recovery]
Resuspend beads in  RNA elution buffer
17 µl
?. [1st mRNA extraction]
Resuspend beads in  RNA elution buffer
17 µl
?. [2nd mRNA recovery]
Warm at  for   in a thermal cycler. After that, cool it quickly on ice for
80 °C
Keep TTT away from AAA
?. [2nd mRNA recovery]
Place 8-strip on magnet rack and transfer supernatant ( ) to a new 8-strip
17 µl
Supernatant here is GOOD for SHO protocol
Can be stored at   if needed
?. [RNA fragmentation & 3-prime adapter cDNA priming]
Mix 3 strand priming with  extracted RNA =   in totalKeep the remaining at )
2.5 µl
7.5 µl
10 µl
-20 °C
?. [RNA fragmentation & 3-prime adapter cDNA priming]
Make 3 strand priming ComponentVolume5X Thermo Scientific RT buffer1.5 μl3-prime priming adapter1 μl
2.5 µl
ComponentVolume5X Thermo Scientific RT buffer1.5 μl3-prime priming adapter1 μl
?. [RNA fragmentation & 3-prime adapter cDNA priming]
of mixed in thermo cycler for RNA fragmentation
10 µl
Fragmentation/Priming program: for DGE (  ,   ,   ,   ,   )
Fragmentation/Priming program: for DGE (  ,   ,   ,   ,   )
?. [RNA fragmentation & 3-prime adapter cDNA priming]
master mix +  fragmented RNA =  mixed solution1st strand master mixComponentvol/rxn5X Thermo Scientific RT buffer1.5 μl0.5M DTT0.3 μlH2O2.2 μl25mM dNTPs0 .5 μlRevertAid RT enzyme0.5 μl
5 µl
10 µl
15 µl
Componentvol/rxn5X Thermo Scientific RT buffer1.5 μl0.5M DTT0.3 μlH2O2.2 μl25mM dNTPs0 .5 μlRevertAid RT enzyme0.5 μl
?. [RNA fragmentation & 3-prime adapter cDNA priming]
Mixed solution in thermo cycler for reverse transcription
Condition:  ,   ,   ,   ,
?. [RNA fragmentation & 3-prime adapter cDNA priming]
Use Ampure beads solution for cDNA purification; Ampure bead solution +  cDNAAmpure bead solutionComponentVolume50 mM EDTA pH 8.05 μlAmpure beads30 μl
35 µl
20 µl
ComponentVolume50 mM EDTA pH 8.05 μlAmpure beads30 μl
?. [RNA fragmentation & 3-prime adapter cDNA priming]
Vortex for  at
0 Room temperature
?. [RNA fragmentation & 3-prime adapter cDNA priming]
Place 8-strip on magnet rack and remove supernatant
?. [RNA fragmentation & 3-prime adapter cDNA priming]
Wash with  ethanol without suspending; Dry the pelletRepeat this process twice
200 µl
Don't dry the pellet too much at 2nd time, otherwise it's hard to elute
Not recommend to store at   at this stage
?. [5-prime adapter sequence addition]
Prepare the master mix in advance during cDNA synthesis, and add the enzyme just before using mater mix/rxn +  suspended cDNA pelletmater mix recipeComponentVolumeH2O3.5 μl10X PolI buffer1 μl250 mM MgCl21 μl25 mM dNTPs0 .25 μlDNA Pol I0 .25 μl
6 µl
4 µl
ComponentVolumeH2O3.5 μl10X PolI buffer1 μl250 mM MgCl21 μl25 mM dNTPs0 .25 μlDNA Pol I0 .25 μl
?. [5-prime adapter sequence addition]
Add  of 10 μM 5-prime adapter directly to the pellet at  to resuspend the bead pellet
4 µl
0 Room temperature
?. [5-prime adapter sequence addition]
Sit at  for
0 Room temperature
?. [5-prime adapter sequence addition]
Ampure bead solution +  cDNA Ampure bead solution recipeComponentVolume50 mM EDTA pH 8.010 μlAmpure beads30 μl
40 µl
10 µl
ComponentVolume50 mM EDTA pH 8.010 μlAmpure beads30 μl
?. [5-prime adapter sequence addition]
Vortex for  at
0 Room temperature
?. [5-prime adapter sequence addition]
Place 8-strip on magnet rack and remove supernatant
?. [5-prime adapter sequence addition]
Wash with  ethanol without suspending; Dry the pelletRepeat this process twice
200 µl
Don't dry the pellet too much at 2nd time, otherwise it's hard to elute
?. [5-prime adapter sequence addition]
Elute the pellet in  of 10 mM Tris pH 8.0; Let it sit for
20 µl
?. [5-prime adapter sequence addition]
Transfer the supernatant to new tubes
?. [Enrichment and adapter extension]
Enrichment master mix +  1 μM ILL-INDEX primer +   cDNAEnrichment master mix recipeComponentVolume2 X KAPA10 μl2 μM PE1 primer1 μl8 μM each EnrichS1 + S2 primers1 μl25mM dNTPs0.2 μl
12.2 µl
2 µl
5.8 µl
ComponentVolume2 X KAPA10 μl2 μM PE1 primer1 μl8 μM each EnrichS1 + S2 primers1 μl25mM dNTPs0.2 μl
?. [Enrichment and adapter extension]
Mixed solution in thermo cycler
, (  ,   ,   ) * 18 cycles,  ,
?. [Final Cleanup]
Ampure beads + of enrichment product; Mix well
24 µl
20 µl
?. [Final Cleanup]
Vortex for  at
0 Room temperature
?. [Final Cleanup]
Place 8-strip on magnet rack and remove supernatant
?. [Final Cleanup]
Wash with  ethanol without suspending; Dry the pelletRepeat this process twice
200 µl
Don't dry the pellet too much at 2nd time, otherwise it's hard to elute
?. [Final Cleanup]
Elute the pellet in  of H2O
12 µl
?. [Final Cleanup]
Use Bioanalyzer for quantification for measuring the concentration of each sample
?. [Library preparation]
Mix equimolar of samples in a 1.5 mL tube
?. [Library preparation]
Add same amount of AMpure XP as the DNA, mix well, and let it stand for
?. [Library preparation]
Place on magnet rack for , remove supernatant and wash twice with  80% EtOH. Dry.
70 µl
?. [Library preparation]
Resuspend the beads by adding  of water, leave them in the magnet rack for Transfer the supernatant to a new 0.5 mL low bind tube.
22 µl
?. [Library preparation]
Quantification with Qbit or Bioanalyzer