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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;"|'''Bar Code Primer 2''' | align="center" style="background:#f0f0f0;"|'''Type of Seq''' | align="center" style="background:#f0f0f0;"|'''Reads''' | align="center" style="background:#f0f0f0;"|'''Library Conc''' |- | BL_totoRNAseq_10ngUHRR_Nov13||UHRR/ERCC||10ng - RNase3 (RIII buffer)/Ambion PAP 10min ATP||TSO.r06||T20VN_PE_R||N2.id1||totoRNAseq|||| |- | ||UHRR/ERCC||10ng - RNase3 (RIII buffer)/Ambion PAP 10min ATP/3'dATP (1:0.1)||TSO.r06||T20VN_PE_R||N2.id2||totoRNAseq|||| |- | ||UHRR/ERCC||10ng - RNase3 (RIII buffer)/Ambion PAP 10min ATP/dATP (1:1)||TSO.r06||T20VN_PE_R||N2.id3||totoRNAseq|||| |- | ||UHRR/ERCC||10ng - RNase3 (RIII buffer)/Affymetrix PAP 10min ATP||TSO.r06||T20VN_PE_R||N2.id4||totoRNAseq|||| |- | ||UHRR/ERCC||10ng - RNase3 (RIII buffer)/Affymetrix PAP 10min ATP/3'dATP (1:0.1)||TSO.r06||T20VN_PE_R||N2.id5||totoRNAseq|||| |- | ||UHRR/ERCC||10ng - RNase3 (RIII buffer)/Affymetrix PAP 10min ATP/dATP (1:1)||TSO.r06||T20VN_PE_R||N2.id6||totoRNAseq|||| |- | ||UHRR/ERCC||10ng - RNase3 (RIII buffer)/Affymetrix PAP 10min ATP then add 3'dATP||TSO.r06||T20VN_PE_R||N2.id7||totoRNAseq|||| |- | ||UHRR/ERCC||10ng - RNase3 (Affy buffer)/Affymetrix PAP 10min ATP then add 3'dATP||TSO.r06||T20VN_PE_R||N2.id8||totoRNAseq|||| |- |} Primer sequences [http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNAseq_Primer_List#totoRNA_5.27_primer_sequence] == Purpose == *Test original totoRNAseq protocol (using RNaseIII buffer for RnaseIII and addition of 5xRT buffer for PAP treatment) without heating to 65C to inactivate enzymes. RNaseIII inactivation by RNase inhibitor addition and PAP limitation by 3'dATP or dATP addition *Testing difference of Ambion PAP and Affymetrix PAP (note that Affymetrix PAP showed significant A tailing of TSO and was completely inhibited by 3'dATP) *Testing Affymetrix PAP in RnaseIII buffer/RT buffer versus Affymetrix buffer (5x stock: 100mM Tris-HCl, pH 7.0, 3.0mM MnCl2, 0.1mM EDTA, 1mM DTT, 500 ΞΌg/ml acetylated BSA, 50% glycerol) Note that the Affymetrix buffer does not have the high salts seen in other PAP buffers and may be more compatible with RNaseIII as well as RT == RNAseIII-Fragmented Library Preparation == :'''RNA''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |UHRR 20ng/ul ||||||||||0.5ul |- |ERCC 1:10E3||||||||||0.2ul |- |1uM T20VN||||||||||0.1ul |- |dH2O||||||||||0.2ul |- |Total||||||||||1ul |- |} :::''Incubate @ 72C 3 min'' :::''Incubate @ 37C 1 min'' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |10x RNaseIII buffer or 5x PAP buffer||||||||||0.2ul or 0.4ul |- |RNase III||||||||||0.1ul |- |dH20||||||||||0.7ul or 0.5ul |- |Total||||||||||2ul |- |} :::''Incubate @ 37C 5 min'' :'''3' Tailing''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |fRNA||||||||||2ul |- |5x RT buffer or 5x Affy PAP Buffer||||||||||0.5ul or 0.4ul |- |1mM ATP (or mix)||||||||||0.5ul |- |2U/ul Ambion PAP or 600U/ul Affymetrix PAP||||||||||0.2ul |- |40U/ul Rnase Inhibitor||||||||||0.2ul |- |dH2O||||||||||0.6ul or 0.7ul |- |Total||||||||||4ul |- |} :::''Incubate @ 37C 10 min'' :::''Add 1ul of 1uM T20VN or 0.5ul 2uM T20VN/0.05uM 3'dATP'' :'''Reverse Transcription''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |PAP RNA ||||||||||5ul (or 4.5ul) |- |5xRT Buffer||||||||||1.5ul (or 2ul) |- |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''' ::#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 :'''TSO on 1/2 RT Reaction''' ::{| {{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'' :'''PCR (PhusionHF)''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |TSO Reaction||||||||||10ul |- |2x Buffer||||||||||25ul |- |10uM P1-STRT||||||||||1ul |- |10uM PCR_R_N2_id||||||||||1ul |- |H2O||||||||||13ul |- |Total||||||||||50ul |- |} ::{| {{table}} |- |98C 30 sec|||||||||| |- |98C 15s -> 58C 20s -> 72C 30s||||||||||6x |- |98C 10s -> 72C 20s||||||||||9x |- |72C, 5min |- |} == Results of Amplification == *Run 5ul on gel: [[File:11132013_PCR1_gel1.jpg]] '''ERCC Amplification''' *Test the ERCC amplification by comparing PCR product with the RT reaction: **PCR product (beads purified and in 10 ul: used 1:5 dilution) **RT reaction (remaining half of RT: used 1:10 dilution) **PCR: ***Sybr 12.5ul ***F primer 0.5ul ***R Primer 0.5ul ***Sample (id1-7) 0.5ul ***dH2O 10.5ul '''PCR Plate format:''' ::{| {{table}} |- |Sample||<u>PCRF/R227</u>||<u>L127/R227</u>||<u>PCRF/R964</u>||<u>L857/R964</u>||<u>PCRF/R227</u>||<u>L127/R227</u>||<u>PCRF/R964</u>||<u>L857/R964</u> |- |Id1||PCR||PCR||PCR||PCR||RT||RT||RT||RT |- |Id2||PCR||PCR||PCR||PCR||RT||RT||RT||RT |- |Id3||PCR||PCR||PCR||PCR||RT||RT||RT||RT |- |Id4||PCR||PCR||PCR||PCR||RT||RT||RT||RT |- |Id5||PCR||PCR||PCR||PCR||RT||RT||RT||RT |- |Id6||PCR||PCR||PCR||PCR||RT||RT||RT||RT |- |Id7||PCR||PCR||PCR||PCR||RT||RT||RT||RT |- |NC||PCR||PCR||PCR||PCR||RT||RT||RT||RT |- |} '''PCR1: ERCC 130 amplification using 5' primer set or 3' primer set:''' [[File:11132013_PCR3_ERCC_1.jpg]] '''PCR2: Amplification using ILMN_PCR_F primer and either the 5' or 3' reverse primer (Note that NC includes RT samples):''' [[File:11132013_PCR3_ERCC_2.jpg]] Results: While the Affymetrix PAP showed higher amplification in the original PCR reaction (P1-STRT/N2.id) and upon reamplification using ILMN_PCR_F/R, the Ambion PAP appears to amplify the ERCC more and showed no real bias between the 5' and 3' ends of the transcript. However, the PCR curve looks more normal for the Affymetrix PAP. Likely the Ambion results are questionable - the curves for the Affymetrix PAP look to be more accurate and reasonable. I can conclude however that the Affymetrix libraries are real and represent amplified transcripts. == Final Library Preparation == *Beads purified twice at (0.8x), re-suspend in 10ul *Re-amplified 0.5ul using ILMN_PCR_F/R and SYBR: [[File:11132013_PCR2_gel1.jpg]] *Combined 2ul of original PCR of libraries 1-7. Ran 1ul on gel: [[File:11132013_PCR1_gel2.jpg]]
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