Blue:RNA-Seq Experiments:10162013
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RNA-Seq Library Information[edit]
Library ID | Samples | Input RNA | TSO Primer | Bar Code Primers | Bar Code Primer 2 | Type of Seq | Reads | Library Conc |
BL_huNu_SingleCell_N2id_81 | Human Nuclei (NeuN+) | Single cell | TSO.r06 | T30.SE.id1-6 | N2.id81 | totoRNAseq | Read1 plus N2 barcode read and Read2 for T20 barcode read | |
BL_huNu_SingleCell_N2id_81 | Human Nuclei (NeuN+) | Single cell | TSO.r06 | T20V.id7-24(except 9&17) | N2.id81 | totoRNAseq | Read1 plus N2 barcode read and Read2 for T20 barcode read | |
BL_huNu_SingleCell_N2id_81 | UHRR | 10pg | TSO.r06 | T20V.id9 | N2.id81 | totoRNAseq | Read1 plus N2 barcode read and Read2 for T20 barcode read | |
BL_huNu_SingleCell_N2id_82 | UHRR | 250pg | TSO.r06 | T20V.id17 | N2.id82 | totoRNAseq | Read1 plus N2 barcode read and Read2 for T20 barcode read |
Plate Layout: Samples and Indexes[edit]
' | 1 | 2 | 3 |
A | NC(1) | UHRR 10pg(9) | UHRR 250pg(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) | 10cells(24) |
Note: SC=Single cell; UHRR=Universal Human Reference RNA
Nuclei Sorted 10152013
Purpose[edit]
- Generate totoRNAseq single cell libraries using modified Smarter TSO (r06) as done in 10012013
RNAseIII-Fragmented Library Preparation[edit]
- Cell Sort
Volume RNA 250pg or Single Nuclei - 1x Lysis Buffer 0.75ul Total 0.75ul
Note: add in UHRR control at this point
- Lysis
Volume RNA 0.75ul 10x RNaseIII Buffer 0.2ul 0.1uM T20V.id 0.1ul dH20 0.2ul (1250pg/ul UHRR for 250pg or 50pg/ul for 10pg) Total 1.25ul
- Incubate @ 72C 3 min
- Incubate @ 37C 1min
- Incubate @ 4C 10min
- RNA FRAGMENTATION
Volume RNA 1.25ul RNase III 0.08ul dH20 0.67ul Total 2ul
- Incubate @ 37C 10 min
- Add 1.1ul dH2O
- Incubate @ 65C 10 min
- 3' Tailing
Volume fRNA 3.1ul 5x RT Buffer 0.5ul 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 4.1ul 10mM dNTP Mix 1ul 0.1uM T20V.ID 0.9ul
- Incubate @ 65C 3 min
- Transfer to Ice
Volume 5xRT Buffer 1.5ul 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
- Remove supernatant and wash 2x with 1ml 80% EtOH
- Air Dry 10min or until cracking first occurs
- Resuspend beads in 10ul dH2O, incubate 2min, transfer to new tube - vacuspin 8 min to bring to 3ul
- TSO
Volume Purified cDNA 3ul 5x RT buffer [MMLV] 2ul 10mM dNTP mix 1ul 100mM DTT 0.25ul 10uM TSO 1ul Betaine (5M) 2ul RNase Inhibitor 0.25ul Smarter MMLV 0.5ul Total 10ul
- Incubate @42C 10min
- Incubate @70C 10min
- PCR (PhusionHF)
Volume TSO Reaction 11ul 2x Buffer 25ul 10uM P1-STRT 1ul 10uM PCR_R_N2_id 1ul H2O 12ul Total 50ul
98C 30 sec 98C 15s -> 58C 20s -> 72C 30s 6x 98C 10s -> 72C 20s 12x 72C, 5min
Results of Amplification[edit]
- Run 5ul on gel:
Final Library Preparation[edit]
- Beads purified twice at (0.8x), re-suspend in 10ul
- Re-Amplified using ILMN_PCR_F/R:
- huNu (~30) id81 = 2ul original PCR
- UHRR (250pg) = 0.5ul original PCR
- NC
- Ran 5ul (of 50ul) on gel:
- Clearly the huNu library did not work...