Blue:RNA-Seq Experiments:09302013: Difference between revisions

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== RNA-Seq Library Information ==
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Library ID'''
| align="center" style="background:#f0f0f0;"|'''Samples'''
| align="center" style="background:#f0f0f0;"|'''Input RNA'''
| align="center" style="background:#f0f0f0;"|'''TSO Primer'''
| align="center" style="background:#f0f0f0;"|'''Bar Code Primers'''
| align="center" style="background:#f0f0f0;"|'''PCR Primers'''
| align="center" style="background:#f0f0f0;"|'''Number of Cycles Amp'''
| align="center" style="background:#f0f0f0;"|'''Type of Seq'''
| align="center" style="background:#f0f0f0;"|'''Reads'''
| align="center" style="background:#f0f0f0;"|'''Library Conc'''
|-
| BL_UHRR_||UHRR/ERCC||1ng - RnaseIII Fragmented 10 min||TSO_r04_N6||T20V.id1||PB_PCR_F/N2_id1||15 Cycles||totoRNAseq||||
|-
| BL_UHRR_||UHRR/ERCC||1ng - RnaseIII Fragmented 5 min||TSO_r04_N6||T20V.id1||PB_PCR_F/N2_id2||15 Cycles||totoRNAseq||||
|-
| BL_UHRR_||UHRR/ERCC||100pg (x2) - RNaseIII Fragmented 10 min||TSO_r04_N6||T20V.id1||PB_PCR_F/N2_id3-4||18 Cycles||totoRNAseq||||
|-
| BL_UHRR_||UHRR/ERCC||100pg (x2) - RNaseIII Fragmented 5 min||TSO_r04_N6||T20V.id1||PB_PCR_F/N2_id5-6||18 Cycles||totoRNAseq||||
|-
| BL_UHRR_||UHRR/ERCC||10pg (x2) - RNaseIII Fragmented 10 min||TSO_r04_N6||T20V.id1||PB_PCR_F/N2_id7-8||21 Cycles||totoRNAseq||||
|-
| BL_UHRR_||UHRR/ERCC||10pg (x2) - RNaseIII Fragmented 5 min||TSO_r04_N6||T20V.id1||PB_PCR_F/N2_id9-10||21 Cycles||totoRNAseq||||
|-
| BL_HuNu_||HuNu/ERCC||1ng - RnaseIII Fragmented 10 min||TSO_r04_N6||T20V.id1||PB_PCR_F/N2_id11||15 Cycles||totoRNAseq||||
|-
| BL_HuNu_||HuNu/ERCC||1ng - RnaseIII Fragmented 5 min||TSO_r04_N6||T20V.id1||PB_PCR_F/N2_id12||15 Cycles||totoRNAseq||||
|-
| BL_HuLCM_||HuLCM/ERCC||1ng - RnaseIII Fragmented 10 min||TSO_r04_N6||T20V.id1||PB_PCR_F/N2_id13||15 Cycles||totoRNAseq||||
|-
| BL_HuLCM_||HuLCM/ERCC||1ng - RnaseIII Fragmented 5 min||TSO_r04_N6||T20V.id1||PB_PCR_F/N2_id14||15 Cycles||totoRNAseq||||
|-
| BL_UHRR_||UHRR/ERCC||1ng - RnaseIII Fragmented 10 min||TSO.r06||T20V.id1||P1-STRT/N2_id15||15 Cycles||totoRNAseq||||
|-
| BL_UHRR_||UHRR/ERCC||1ng - RnaseIII Fragmented 5 min||TSO.r06||T20V.id1||P1-STRT/N2_id16||15 Cycles||totoRNAseq||||
|-
| BL_UHRR_||UHRR/ERCC||100pg (x2) - RNaseIII Fragmented 10 min||TSO.r06||T20V.id1||P1-STRT/N2_id17-18||18 Cycles||totoRNAseq||||
|-
| BL_UHRR_||UHRR/ERCC||100pg (x2) - RNaseIII Fragmented 5 min||TSO.r06||T20V.id1||P1-STRT/N2_id19-20||18 Cycles||totoRNAseq||||
|-
| BL_UHRR_||UHRR/ERCC||10pg (x2) - RNaseIII Fragmented 10 min||TSO.r06||T20V.id1||P1-STRT/N2_id21-22||21 Cycles||totoRNAseq||||
|-
| BL_UHRR_||UHRR/ERCC||10pg (x2) - RNaseIII Fragmented 5 min||TSO.r06||T20V.id1||P1-STRT/N2_id23-24||21 Cycles||totoRNAseq||||
|-
| BL_HuNu_||HuNu/ERCC||1ng - RnaseIII Fragmented 10 min||TSO.r06||T20V.id1||P1-STRT/N2_id25||15 Cycles||totoRNAseq||||
|-
| BL_HuNu_||HuNu/ERCC||1ng - RnaseIII Fragmented 5 min||TSO.r06||T20V.id1||P1-STRT/N2_id26||15 Cycles||totoRNAseq||||
|-
| BL_HuLCM_||HuLCM/ERCC||1ng - RnaseIII Fragmented 10 min||TSO.r06||T20V.id1||P1-STRT/N2_id27||15 Cycles||totoRNAseq||||
|-
| BL_HuLCM_||HuLCM/ERCC||1ng - RnaseIII Fragmented 5 min||TSO.r06||T20V.id1||P1-STRT/N2_id28||15 Cycles||totoRNAseq||||
|-
|}
[http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNAseq_Primer_List Primer List]
UHRR=Universal Human Reference RNA; ERCC=spike in RNA; HuNu = RNA from Human Pyramidal Nuclei sorted 09-23-13; HuLCM = RNA from bulk Human Pyramidal LCM sample
== Purpose ==
*Generate totoRNAseq libraries using modified Smarter TSO (r06) and modified r04 TSO to compare efficiencies
*Compare TSO efficiencies over different RNA amounts and between UHRR and human pyramidal RNA generated from sorted nuclei or LCM
*Compare RNAseIII fragmentation times: full (10min) or partial (5min)
== RNAseIII-Fragmented Library Preparation ==
:'''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||||||||||0.2ul
|-
|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'''
::{| {{table}}
|-
|||||||||||<u>Volume</u>
|-
|RNA||||||||||1.25ul
|-
|RNase III||||||||||0.08ul
|-
|dH20||||||||||0.67ul
|-
|Total||||||||||2ul
|-
|}
:::''Incubate @ 37C 5-10 min''
:::''Add 1.1ul dH2O
:::''Incubate @ 65C 10 min''
:'''3' Tailing'''
::{| {{table}}
|-
|||||||||||<u>Volume</u>
|-
|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'''
::{| {{table}}
|-
|||||||||||<u>Volume</u>
|-
|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''
::{| {{table}}
|-
|||||||||||<u>Volume</u>
|-
|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
::#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 5 min to bring to 3ul
:'''TSO'''
::{| {{table}}
|-
|||||||||||<u>Volume</u>
|-
|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)'''
::{| {{table}}
|-
|||||||||||<u>Volume</u>
|-
|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
|-
|}
::{| {{table}}
|-
|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 ==

Revision as of 22:03, 26 September 2013