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 = Human Pyramidal Nuclei sorted 09-23-13
== Purpose ==
== Purpose ==


*Generate totoRNAseq libraries using several TSO to compare efficiencies
*mNPCs from E12.5 and E10.5 mice were isolated by Yun
*Compare TSO efficiencies over different RNA amounts and between UHRR and human pyramidal nuclei
*Sorting of mNPCs (mixed population of cell types) was based on PI negative staining


== RNAseIII-Fragmented Library Preparation ==
== Smart-Seq Sort Buffer: ==




:'''RNA PREPARATION'''
{| {{table}}
 
| align="center" style="background:#f0f0f0;"|'''Component'''
::{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Stock'''
|-
| align="center" style="background:#f0f0f0;"|'''Final (1x)'''
|||||||||||<u>Volume</u>
| align="center" style="background:#f0f0f0;"|'''Volume'''
|-
|2ng/ul, 200ng/ul, 20ng/ul UHRR||||||||||0.5ul
|-
|ERCC 1:10E4, 1:10E5, 1:10E6||||||||||0.2ul
|-
|-
|10x RNaseIII Buffer||||||||||0.2ul
| Clontech Dilution Buffer|| || ||820ul
|-
|-
|1uM or 0.1uM (10-100pg) T20V.id1 ||||||||||0.1ul
| Rnase Inhibitor||40U/ul||1U/ul||21.25ul
|-
|-
|dH20||||||||||0.25ul
| ERCC spike-in RNA||1:10^5||||8.5ul
|-
|-
|Total||||||||||1.25ul
| total||||||850ul
|-
|-
|
|}
|}


:::''Incubate @ 72C 3 min''
:::''Incubate @ 37C 1min''


::'''Note: using 2ul per well for 4 plates (two E12.5 and two E10.5)


:'''RNA FRAGMENTATION'''
== Universal Sort Buffer: ==


::{| {{table}}
|-
|||||||||||<u>Volume</u>
|-
|RNA||||||||||1.25ul
|-
|RNase III||||||||||0.08ul
|-
|dH20||||||||||0.67ul
|-
|Total||||||||||2ul
|-
|}


 
{| {{table}}
:::''Incubate @ 37C 5-10 min''
| align="center" style="background:#f0f0f0;"|'''Component'''
 
| align="center" style="background:#f0f0f0;"|'''Stock'''
:::''Add 1.1ul dH2O
| align="center" style="background:#f0f0f0;"|'''Final (1x)'''
 
| align="center" style="background:#f0f0f0;"|'''Volume'''
:::''Incubate @ 65C 10 min''
 
 
 
:'''3' Tailing'''
 
::{| {{table}}
|-
|-
|||||||||||<u>Volume</u>
| DEPC Water|| || ||469ul
|-
|-
|fRNA||||||||||3.1ul
| 10% Triton-X100|| || ||5ul
|-
|-
|5x RT Buffer||||||||||0.5ul
| Rnase Inhibitor||40U/ul||1U/ul||12.5ul
|-
|-
|1mM ATP||||||||||0.25ul
| ERCC spike-in RNA||1:10^5||||13.25ul
|-
|-
|0.5U/ul PAP (1:10d in H2O)||||||||||0.25ul
| total||||||500ul
|-
|-
|
|}
|}




:::''Incubate @ 37C 5 min''
::'''Note: using 0.75ul per well for 5 plates (three for E12.5, two for E10.5) and bubbling just prior to sort
:::''Transfer to Ice''


== Plate Layout: ==




:'''Reverse Transcription'''
{| {{table}}
 
| align="center" style="background:#f0f0f0;"|''''''
::{| {{table}}
| align="center" style="background:#f0f0f0;"|'''1'''
|-
| align="center" style="background:#f0f0f0;"|'''2'''
|||||||||||<u>Volume</u>
| align="center" style="background:#f0f0f0;"|'''3'''
|-
| align="center" style="background:#f0f0f0;"|'''4'''
|PAP RNA ||||||||||4.1ul
| align="center" style="background:#f0f0f0;"|'''5'''
|-
| align="center" style="background:#f0f0f0;"|'''6'''
|10mM dNTP Mix||||||||||1ul
| align="center" style="background:#f0f0f0;"|'''7'''
|-
| align="center" style="background:#f0f0f0;"|'''8'''
|1uM or 0.1uM (10-100pg) T20V.ID ||||||||||0.9ul
| align="center" style="background:#f0f0f0;"|'''9'''
| align="center" style="background:#f0f0f0;"|'''10'''
| align="center" style="background:#f0f0f0;"|'''11'''
| align="center" style="background:#f0f0f0;"|'''12'''
|-
|-
|}
| A||Sc(1)||UHRR(9)||NC(17)||Sc(25)||UHRR(33)||NC(41)||Sc(49)||UHRR(57)||NC(65)||Sc(73)||UHRR(81)||NC(89)
 
 
 
:::''Incubate @ 65C 3 min''
:::''Transfer to Ice''
 
 
 
::{| {{table}}
|-
|-
|||||||||||<u>Volume</u>
| B||Sc(2)||Sc(10)||Sc(18)||Sc(26)||Sc(34)||Sc(42)||Sc(50)||Sc(58)||Sc(66)||Sc(74)||Sc(82)||Sc(90)
|-
|-
|5xRT Buffer||||||||||1.5ul
| C||Sc(3)||Sc(11)||Sc(19)||Sc(27)||Sc(35)||Sc(43)||Sc(51)||Sc(59)||Sc(67)||Sc(75)||Sc(83)||Sc(91)
|-
|-
|100mM DTT||||||||||0.17ul
| D||Sc(4)||Sc(12)||Sc(20)||Sc(28)||Sc(36)||Sc(44)||Sc(52)||Sc(60)||Sc(68)||Sc(76)||Sc(84)||Sc(92)
|-
|-
|Betaine (5M)||||||||||2ul
| E||Sc(5)||Sc(13)||Sc(21)||Sc(29)||Sc(37)||Sc(45)||Sc(53)||Sc(61)||Sc(69)||Sc(77)||Sc(85)||Sc(93)
|-
|-
|RNase Inhibitor (40U/ul)||||||||||0.1ul
| F||Sc(6)||Sc(14)||Sc(22)||Sc(30)||Sc(38)||Sc(46)||Sc(54)||Sc(62)||Sc(70)||Sc(78)||Sc(86)||Sc(94)
|-
|-
|Smarter MMLV (200U/ul)||||||||||0.23ul
| G||Sc(7)||Sc(15)||Sc(23)||Sc(31)||Sc(39)||Sc(47)||Sc(55)||Sc(63)||Sc(71)||Sc(79)||Sc(87)||Sc(95)
|-
|-
|Total||||||||||10ul
| H||Sc(8)||Sc(16)||10cell(24)||Sc(32)||Sc(40)||10cell(48)||Sc(56)||Sc(64)||10cell(72)||Sc(80)||Sc(88)||10cell(96)
|-
|-
|
|}
|}




:::''Incubate @ 42C 30 min''
Sc=Single cell; (#)=Indx
 
 
:'''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 ==
'''Notes:'''
*Sort was done on the FACS ARIA II
*Sort files: [[File:9-30-13_E10.5_mNPC_snigle_cell_sort.pdf]]

Latest revision as of 21:00, 30 September 2013

Purpose[edit]

  • mNPCs from E12.5 and E10.5 mice were isolated by Yun
  • Sorting of mNPCs (mixed population of cell types) was based on PI negative staining

Smart-Seq Sort Buffer:[edit]

Component Stock Final (1x) Volume
Clontech Dilution Buffer 820ul
Rnase Inhibitor 40U/ul 1U/ul 21.25ul
ERCC spike-in RNA 1:10^5 8.5ul
total 850ul


Note: using 2ul per well for 4 plates (two E12.5 and two E10.5)

Universal Sort Buffer:[edit]

Component Stock Final (1x) Volume
DEPC Water 469ul
10% Triton-X100 5ul
Rnase Inhibitor 40U/ul 1U/ul 12.5ul
ERCC spike-in RNA 1:10^5 13.25ul
total 500ul


Note: using 0.75ul per well for 5 plates (three for E12.5, two for E10.5) and bubbling just prior to sort

Plate Layout:[edit]

' 1 2 3 4 5 6 7 8 9 10 11 12
A Sc(1) UHRR(9) NC(17) Sc(25) UHRR(33) NC(41) Sc(49) UHRR(57) NC(65) Sc(73) UHRR(81) NC(89)
B Sc(2) Sc(10) Sc(18) Sc(26) Sc(34) Sc(42) Sc(50) Sc(58) Sc(66) Sc(74) Sc(82) Sc(90)
C Sc(3) Sc(11) Sc(19) Sc(27) Sc(35) Sc(43) Sc(51) Sc(59) Sc(67) Sc(75) Sc(83) Sc(91)
D Sc(4) Sc(12) Sc(20) Sc(28) Sc(36) Sc(44) Sc(52) Sc(60) Sc(68) Sc(76) Sc(84) Sc(92)
E Sc(5) Sc(13) Sc(21) Sc(29) Sc(37) Sc(45) Sc(53) Sc(61) Sc(69) Sc(77) Sc(85) Sc(93)
F Sc(6) Sc(14) Sc(22) Sc(30) Sc(38) Sc(46) Sc(54) Sc(62) Sc(70) Sc(78) Sc(86) Sc(94)
G Sc(7) Sc(15) Sc(23) Sc(31) Sc(39) Sc(47) Sc(55) Sc(63) Sc(71) Sc(79) Sc(87) Sc(95)
H Sc(8) Sc(16) 10cell(24) Sc(32) Sc(40) 10cell(48) Sc(56) Sc(64) 10cell(72) Sc(80) Sc(88) 10cell(96)


Sc=Single cell; (#)=Indx


Notes: