Blue:RNA-Seq Experiments:01292014: Difference between revisions

From ZhangLabWiki
Jump to navigation Jump to search
>B1lake
>B1lake
Line 217: Line 217:
*Run 5ul on gel:
*Run 5ul on gel:


[[File:01292014_PCR1_Gel1.jpg|400px]]
[[File:01292014_PCR1_Gel1.jpg|300px]]





Revision as of 01:24, 31 January 2014

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_totoRNAseq_10pgUHRR_Jan29 UHRR/ERCC 10x10pg - 0.05U RNase3 1min/PAP 4min/RT 42C 30min TSO.r06 T20VN_id1-10 N2.id1 totoRNAseq
hNPC/ERCC 10xSC - 0.05U RNase3 1min/PAP 4min/RT 37C 60min TSO.r06 T20VN_id1-10 N2.id2 totoRNAseq
hNPC/ERCC 10xSC - 0.05U RNase3 1min/PAP 4min/RT 37C 60min TSO.r06 T20VN_id1-10 N2.id3 totoRNAseq
UHRR/ERCC 4x1ng - 0.1U RNase3 1min/PAP 10min/RT 42C 30min TSO.r06 T20VN_r01 N2.id4-8 totoRNAseq

Note: hNPCs from [1] - excluding the 10pg control, 0 cell controls and 10 cell control. Combined 10 cells per N2 index for ~100pg total RNA

Primer sequences [2]

Purpose

  • Test optimized RNase III and PAP conditions on 10pg RNA based on 01222014
  • 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

RNA
Volume
2ng/ul or 0.02ng/ul or NPC 0.5ul
1:104 or 1:10E6 ERCC (UHRR only) 0.2ul
0.1uM T20VN 0.1ul
dH2O 0.2ul
Total 1ul


Incubate @ 72C 3 min
Incubate @ 37C 1 min
Incubate @ 4C 10 min


Volume
10x RNaseIII buffer 0.2ul
RNase III 0.1ul or 0.05ul
dH20 0.7ul or 0.75ul
Total 2ul


Incubate @ 37C 1 min


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 or 10 min
Add 1ul of 0.1uM T20VN.id


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 or 37C for 60 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 ~8min 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 9x (1ng) or 12x (100pg)
72C, 5min


Results of Amplification

  • Run 5ul on gel:

File:01292014 PCR1 Gel1.jpg


Results:

Final Library Preparation

  • 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.


  • Results: