Blue:RNA-Seq Experiments:01132014
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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_totoRNAseq_10pgUHRR_Jan13 | UHRR/ERCC | 10pg - RNase3 15s/Affymetrix PAP 4min | TSO.r06 | T20VN_id1-10 | N2.id15 | totoRNAseq | ||
hNPC | SC - RNase3 15s/Affymetrix PAP 4min | TSO.r06 | T20VN_id1-8,10-12 | N2.id16 | totoRNAseq | |||
hNPC | SC - RNase3 15s/Affymetrix PAP 4min | TSO.r06 | T20VN_id13-23 | N2.id17 | totoRNAseq | |||
hNPC | 10 cells - RNase3 15s/Affymetrix PAP 4min | TSO.r06 | T20VN_id24 | N2.id18 | totoRNAseq |
Note: hNPCs from [1] - excluding the 10pg control (id9) - combined for 10 cells per N2 index (~100pg total)
Primer sequences [2]
Purpose[edit]
- Test optimized RNase III and PAP conditions on 10pg RNA based on 01082014
- Use ERCCs to determine level of transcript amplification between libraries
- Combine libraries prior to TSO and amplification to reduce variability between samples and amplification cycle number
- Compare with single cell sorted samples ([3])
RNAseIII-Fragmented Library Preparation[edit]
- RNA
Volume 0.1ng/ul (for 10pg UHRR only) 0.1ul 1:10E6 (for 10pg UHRR only) 0.2ul 0.1uM T20VN 0.1ul dH2O 0.6ul or 0.4ul for hNPCs Total 1ul (0.5ul for hNPCs)
- Incubate @ 72C 3 min
- Incubate @ 37C 1 min
Volume 10x RNaseIII buffer 0.2ul RNase III 0.1ul dH20 0.7ul Total 2ul
- Incubate @ 37C 15s-20s
- 3' Tailing
Volume fRNA 2ul 5x RT buffer 0.5ul 1mM ATP 0.5ul 600U/ul Affymetrix PAP 0.2ul 40U/ul Rnase Inhibitor 0.2ul dH2O 0.6ul Total 4ul
- Incubate @ 37C 4 min
- Add 1ul of 0.1uM T20VN
- Reverse Transcription
Volume PAP RNA 5ul 5xRT Buffer 1.5ul 10mM dNTP Mix 1ul 100mM DTT 0.25ul Betaine (5M) 2ul Smarter MMLV (200U/ul) 0.25ul Total 10ul
- Incubate @ 42C 30 min
- Beads Purification
- Add 15ul beads per well
- Allow to bind 15 min, combine wells
- Remove supernatant and wash 2x with 1ml 80% EtOH
- Air Dry 10min or until cracking first occurs
- Resuspend beads in 6ul dH2O, incubate 2min, transfer to new tube; vacuspin to 3ul
- TSO Reaction
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 10ul 2x Buffer 25ul 10uM P1-STRT 1ul 10uM PCR_R_N2_id 1ul H2O 13ul Total 50ul
98C 30 sec 98C 15s -> 58C 20s -> 72C 30s 6x 98C 10s -> 72C 20s 12x (100pg) 72C, 5min
Results of Amplification[edit]
- Run 5ul on gel:
Results: 10 x 10pg UHRR and 10 x 10 hNPCs show similar amplification levels. 10 sorted hNPC cells (single well) shows less amplification - possibly due to the lower contribution of ERCCs and poorer lysis efficiency
Final Library Preparation[edit]
- Beads purified twice at (0.8x), re-suspended in 10ul
- PCR on 0.5ul each library using ILMN_PCR_F and either ERCC130R227 or ERCC130R964.
File:01132014 ERCC130 R227.jpgFile:01132014 ERCC130 R964.jpg
- Results: ERCC130_R227 (5') PCR does not show much difference between samples - id18 had only a single well of ERCC and should be 10 fold less than the others... this does not make sense. ERCC_R964 (3'), however, does show the expected amplification distributions and indicates that the individual 10pg samples combined (100pg for 18 cycles) are similarly amplified as 1ng (for 15 cycles - exp 01082014). hNPC levels were also amplified similarly to the 10pg UHRR samples - sequencing would be needed to confirm and determine consistency between individual 10pg or hNPC single cell libraries.