Blue:RNA-Seq Experiments:08282013: Difference between revisions
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Note: SC=Single cell; UHRR=Universal Human Reference RNA | Note: SC=Single cell => N5_HSC_AIT2 CD34+GFP+; UHRR=Universal Human Reference RNA | ||
Cells Sorted [http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNA-Seq_Experiments:08052013 08052013] | Cells Sorted [http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNA-Seq_Experiments:08052013 08052013] |
Revision as of 22:16, 28 August 2013
RNA-Seq Library Information
Library ID | Samples | Input RNA | TSO Primer | Bar Code Primers | Bar Code Primer 2 | Type of Seq | Reads | Library Conc |
BL_HSC_SingleCell_N2id89_0828 | huES8 | Single cell | TSO.r04 | T20V.id1-24 | N2.id89 | totoRNAseq | Read1 plus N2 barcode read and Read2 for T20 barcode read | |
UHRR | 250pg | TSO.r04 | T20V.id1 | N2.id90 | ||||
UHRR | 250pg | TSO.r04 | T20V.id2 | N2.id91 |
Plate Layout: Samples and Indexes
' | 1 | 2 | 3 |
A | 10cell(1) | 10cell(9) | Sc(17) |
B | Sc(2) | Sc(10) | Sc(18) |
C | Sc(3) | Sc(11) | Sc(19) |
D | Sc(4) | Sc(12) | Sc(20) |
E | Sc(5) | Sc(13) | Sc(21) |
F | Sc(6) | Sc(14) | Sc(22) |
G | Sc(7) | Sc(15) | Sc(23) |
H | Sc(8) | Sc(16) | Sc(24) |
Note: SC=Single cell => N5_HSC_AIT2 CD34+GFP+; UHRR=Universal Human Reference RNA
Cells Sorted 08052013
Purpose
- Try Single cell RNAseIII/TSO totoRNAseq protocol on Zhili's HSC sorted cells
- Cells are in hiMg based buffer, however RNaseIII protocol looks more promising (08202013)
- Test RNaseIII adaptation using hiMg RT sort buffer
Library Preparation (totoRNAseq):
- Cell Sort
Volume RNA 250pg or Single Cells - 2.5x hiMgRT/Lysis Buffer 2ul Total 2ul
Note: add in UHRR control at this point
- Cell Lysis
Volume RNA 2ul 0.1uM T20V.id 0.1ul dH20 0.45ul Total 2.55ul
- Incubate @ 72C 3 min
- Incubate @ 37C 1min
- Incubate @ 4C 10min
- RNA FRAGMENTATION
Volume RNA 2.55ul RNase III 0.08ul dH20 0.47ul Total 3.1ul
- Incubate @ 37C 10 min
- Add 1ul dH2O
- Incubate @ 65C 10 min
- 3' Tailing
Volume PNK_fRNA 4.1ul 1mM ATP 0.25ul 0.5U/ul PAP (1:10d in H2O) 0.25ul
- Incubate @ 37C 5 min
- Transfer to Ice
- Reverse Transcription
Volume PAP RNA 4.6ul 10mM dNTP Mix 1ul 0.1uM T20V.ID 0.9ul
- Incubate @ 65C 3 min
- Transfer to Ice
Volume 5xRT Buffer 1ul 100mM DTT 0.17ul Betaine (5M) 2ul RNase Inhibitor (40U/ul) 0.1ul Smarter MMLV (200U/ul) 0.23ul Total 10ul
- Incubate @ 42C 30 min
- Beads Purification
- Add 15ul beads per well
- Allow to bind 15 min
- Combine all odd or even column wells
- Remove supernatant and wash 2x with 1ml 80% EtOH
- Air Dry 10min or until cracking first occurs
- Resuspend beads in 20ul dH2O, incubate 2min, transfer to new tube
- Vacuspin room temperature for 10+min to bring volume to 6ul (TSO) or 7.7ul (IVT)
TSO
- TSO (x2)
Volume Purified cDNA 3ul 5x RT buffer [MMLV] 2ul 10mM dNTP mix 1ul 100mM DTT 0.25ul 10uM TSO.r04 1ul Betaine (5M) 2ul RNase Inhibitor 0.25ul Smarter MMLV 0.5ul Total 10ul
- Incubate @42C 10min
- Incubate @70C 10min
- USER
- Add 1ul and incubate at 37C for 30 min
- PCR (PhusionHF)
Volume TSO Reaction 11ul 2x Buffer 25ul 10uM PB_PCR_F 1ul 10uM PCR_N2_idX 1ul H2O 12ul Total 50ul
98C 30 sec 98C 15s -> 58C 20s -> 72C 30s 6x 98C 10s -> 72C 20s 15x 72C, 5min
- Ampure Beads Purify (0.8x)
- Resuspend in 50ul
- Ampure Beads Purify (0.8x)
- Resuspend in 10ul
- Run 1ul on gel
Results of Amplification
- Ran 1ul on gel:
Final Library Preparation
- Re-Amplified 1ul using Sybr-Green and ILMN_PCR_F/R
Volume Library 1ul 2xSybr Buffer 25ul 10uM ILMN_PCR_F 1ul 10uM ILMN_PCR_R 1ul dH2O 22ul Total 50ul
- 95C 30sec
- 95C 10s -> 60C 20s -> 72C 30s => 5X
- 72C, 5min
- Ampure Beads purified (0.8x)
- Resuspended in 10ul and ran 1 ul on gel: