Blue:RNA-Seq Experiments:01132014

From ZhangLabWiki
Jump to navigation Jump to search

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
  1. Add 15ul beads per well
  2. Allow to bind 15 min, combine wells
  3. Remove supernatant and wash 2x with 1ml 80% EtOH
  4. Air Dry 10min or until cracking first occurs
  5. 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:

File:01132014 PCR1 Gel1.jpg


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.