Blue:RNA-Seq Experiments:smarter test: Difference between revisions

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>B1lake
>B1lake
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|98C 10s -> 72C 20s||||||||||9x (1ng), 12x (100pg), 18x (10pg)
|98C 10s -> 72C 20s||||||||||9x (1ng), 12x (100pg), 18x (10pg)
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|72C, 5min
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== RNAseIII-Fragmented Library Preparation (1-Step) ==
:'''RNA PREPARATION'''
::{| {{table}}
|-
|||||||||||<u>Volume</u>
|-
|2ng/ul, 200ng/ul, 20ng/ul UHRR||||||||||0.5ul
|-
|ERCC 1:10E4, 1:10E5, 1:10E6||||||||||0.2ul
|-
|10x RNaseIII Buffer||||||||||0.15ul
|-
|1uM T20V.id1 ||||||||||0.1ul
|-
|Total||||||||||0.95ul
|-
|}
:::''Incubate @ 72C 3 min''
:::''Incubate @ 37C 1min''
:'''RNA FRAGMENTATION'''
::{| {{table}}
|-
|||||||||||<u>Volume</u>
|-
|RNA||||||||||0.95ul
|-
|RNase III||||||||||0.08ul
|-
|dH20||||||||||0.47ul
|-
|Total||||||||||1.5ul
|-
|}
:::''Incubate @ 37C 10 min''
:::''Add 0.6ul dH2O
:::''Incubate @ 65C 10 min''
:'''3' Tailing'''
::{| {{table}}
|-
|||||||||||<u>Volume</u>
|-
|fRNA||||||||||2.1ul
|-
|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'''
::{| {{table}}
|-
|||||||||||<u>Volume</u>
|-
|PAP RNA ||||||||||3.1ul
|-
|10mM dNTP Mix||||||||||1ul
|-
|1uM T20V.ID ||||||||||0.9ul
|-
|}
:::''Incubate @ 65C 3 min''
:::''Transfer to Ice''
::{| {{table}}
|-
|||||||||||<u>Volume</u>
|-
|5xRT Buffer||||||||||1.5ul
|-
|100mM DTT||||||||||0.17ul
|-
|Betaine (5M)||||||||||2ul
|-
|TSO.r03 (10uM)||||||||||1ul
|-
|RNase Inhibitor (40U/ul)||||||||||0.1ul
|-
|Smarter MMLV (200U/ul)||||||||||0.23ul
|-
|Total||||||||||10ul
|-
|}
:::''Incubate @42C 30 min''
:::''Incubate @70C 10 min''
:'''USER'''
::''Add 1ul and incubate at 37C for 30 min''
:'''PCR (PhusionHF)'''
::{| {{table}}
|-
|||||||||||<u>Volume</u>
|-
|TSO Reaction||||||||||11ul
|-
|2x Buffer||||||||||25ul
|-
|10uM PB_PCR_F||||||||||1ul
|-
|10uM PCR_R_N2_id ||||||||||1ul
|-
|H2O||||||||||12ul
|-
|Total||||||||||50ul
|-
|}
::{| {{table}}
|-
|98C 30 sec||||||||||
|-
|98C 15s -> 58C 20s -> 72C 30s||||||||||6x
|-
|98C 10s -> 72C 20s||||||||||6x (1ng), 9x (100pg), 12x (10pg)
|-
|-
|72C, 5min
|72C, 5min

Revision as of 00:13, 26 September 2013

RNA-Seq Library Information

Library ID Samples Input RNA TSO Primer Bar Code Primers Bar Code Primer 2 Number of Cycles Amp Type of Seq Reads Library Conc
BL_UHRR_ UHRR/ERCC 1ng - RnaseIII Fragmented Smarter TSO (In-house) T20V.id N2_id1 15 Cycles totoRNAseq
BL_UHRR_ UHRR/ERCC 100pg (x2) - RNaseIII Fragmented Smarter TSO (In-house) T20V.id N2_id2-3 18 Cycles totoRNAseq
BL_UHRR_ UHRR/ERCC 10pg (x2) - RNaseIII Fragmented Smarter TSO (In-house) T20V.id N2_id4-5 21 Cycles totoRNAseq
BL_HuNu_ HuNu/ERCC 1ng - RnaseIII Fragmented 10 min Smarter TSO (In-house) T20V.id N2_id6 15 Cycles totoRNAseq
BL_HuNu_ HuNu/ERCC 1ng - RnaseIII Fragmented 5 min Smarter TSO (In-house) T20V.id N2_id7 15 Cycles totoRNAseq
BL_UHRR_ UHRR/ERCC 1ng - RnaseIII Fragmented r06 TSO (In-house) T20V.id N2_id8 15 Cycles totoRNAseq
BL_UHRR_ UHRR/ERCC 100pg (x2) - RNaseIII Fragmented r06 TSO (In-house) T20V.id N2_id9-10 18 Cycles totoRNAseq
BL_UHRR_ UHRR/ERCC 10pg - (x2) RNaseIII Fragmented r06 TSO (In-house) T20V.id N2_id11-12 21 Cycles totoRNAseq
BL_HuNu_ HuNu/ERCC 1ng - RnaseIII Fragmented 10 min r06 TSO (In-house) T20V.id N2_id13 15 Cycles totoRNAseq
BL_HuNu_ HuNu/ERCC 1ng - RnaseIII Fragmented 5 min r06 TSO (In-house) T20V.id N2_id14 15 Cycles totoRNAseq
BL_UHRR_ UHRR/ERCC 1ng - RnaseIII Fragmented r07 TSO (In-house) T20V.id N2_id15 15 Cycles totoRNAseq
BL_UHRR_ UHRR/ERCC 100pg (x2) - RNaseIII Fragmented r07 TSO (In-house) T20V.id N2_id16-17 18 Cycles totoRNAseq
BL_UHRR_ UHRR/ERCC 10pg (x2) - RNaseIII Fragmented r07 TSO (In-house) T20V.id N2_id18-19 21 Cycles totoRNAseq
BL_HuNu_ HuNu/ERCC 1ng - RnaseIII Fragmented 10 min r07 TSO (In-house) T20V.id N2_id20 15 Cycles totoRNAseq
BL_HuNu_ HuNu/ERCC 1ng - RnaseIII Fragmented 10 min r07 TSO (In-house) T20V.id N2_id21 15 Cycles totoRNAseq


UHRR=Universal Human Reference RNA; ERCC=spike in RNA; HuNu = Human Pyramidal Nuclei sorted 09-23-13

Purpose

  • Generate totoRNAseq libraries using several TSO to compare efficiencies
  • Compare TSO efficiencies over different RNA amounts and between UHRR and human pyramidal nuclei

RNAseIII-Fragmented Library Preparation

RNA PREPARATION
Volume
2ng/ul, 200ng/ul, 20ng/ul UHRR 0.5ul
ERCC 1:10E4, 1:10E5, 1:10E6 0.2ul
10x RNaseIII Buffer 0.2ul
1uM or 0.1uM (10-100pg) T20V.id1 0.1ul
dH20 0.25ul
Total 1.25ul
Incubate @ 72C 3 min
Incubate @ 37C 1min


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
1uM or 0.1uM (10-100pg) 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
  1. Add 15ul beads per well
  2. Allow to bind 15 min
  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 5 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


USER
Add 1ul and incubate at 37C for 30 min


PCR (PhusionHF)
Volume
TSO Reaction 11ul
2x Buffer 25ul
10uM P1-STRT (Smarter TSO) or PB_PCR_F 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 9x (1ng), 12x (100pg), 18x (10pg)
72C, 5min

Results of Amplification

  • Ampure Beads Purify (0.8x)
  • Resuspend in 10ul
  • Run 1ul on gel

Final Library Preparation