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Blue:RNA-Seq Experiments:09182013
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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_UHRR_||UHRR/ERCC||1ng - Unfragmented||STRT_V2_3 (In-house)||STRT_V3_T30 (In-house)||||Smarter||Read 1 and N2 barcode read|| |- | BL_UHRR_||UHRR/ERCC||100pg - Unfragmented||STRT_V2_3 (In-house)||STRT_V3_T30 (In-house)||||Smarter|||| |- | BL_UHRR_||UHRR/ERCC||10pg - Unfragmented||STRT_V2_3 (In-house)||STRT_V3_T30 (In-house)||||Smarter|||| |- | BL_UHRR_||UHRR/ERCC||1ng - Unfragmented-PolyA||STRT_V2_3 (In-house)||STRT_V3_T30 (In-house)||||Smarter Full-length totoRNAseq|||| |- | BL_UHRR_||UHRR/ERCC||100pg - Unfragmented-PolyA||STRT_V2_3 (In-house)||STRT_V3_T30 (In-house)||||Smarter Full-length totoRNAseq|||| |- | BL_UHRR_||UHRR/ERCC||10pg - Unfragmented-PolyA||STRT_V2_3 (In-house)||STRT_V3_T30 (In-house)||||Smarter Full-length totoRNAseq|||| |- | BL_UHRR_||UHRR/ERCC||1ng - RnaseIII Fragmented||STRT_V2_3 (In-house)||STRT_V3_T30 (In-house)||||Smarter totoRNAseq|||| |- | BL_UHRR_||UHRR/ERCC||100pg - RNaseIII Fragmented||STRT_V2_3 (In-house)||STRT_V3_T30 (In-house)||||Smarter totoRNAseq|||| |- | BL_UHRR_||UHRR/ERCC||10pg - RNaseIII Fragmented||STRT_V2_3 (In-house)||STRT_V3_T30 (In-house)||||Smarter totoRNAseq|||| |- | BL_UHRR_||UHRR/ERCC||1ng - RnaseIII Fragmented||STRT_V2_3 (In-house)||T20V.id||N2_id9||totoRNAseq|||| |- | BL_UHRR_||UHRR/ERCC||100pg - RNaseIII Fragmented||STRT_V2_3 (In-house)||T20V.id||N2_id10||totoRNAseq|||| |- | BL_UHRR_||UHRR/ERCC||10pg - RNaseIII Fragmented||STRT_V2_3 (In-house)||T20V.id||N2_id11||totoRNAseq|||| |- | BL_UHRR_||UHRR/ERCC||1ng - RNaseIII Fragmented - 1 step||TSO_r03||T20V.id||N2_id12||totoRNAseq|| |- | BL_UHRR_||UHRR/ERCC||100pg - RNaseIII Fragmented - 1 step||TSO_r03||T20V.id||N2_id13||totoRNAseq|| |- | BL_UHRR_||UHRR/ERCC||10pg - RNaseIII Fragmented - 1 step||TSO_r03||T20V.id||N2_id14||totoRNAseq|| |- |} == Purpose == *Use variations of our protocol to evaluate the quality of library generation using Smart primers (kit or in-house) using ERCC QC assay *Examine efficiency of Smarter protocol (unfragmented) compared to Poly-A tailing (unfragmented) and fragmentation using ERCC QC assay *Examine efficiency of the modified TSO r03 (resistant to Poly-A tailing) in a single step totoRNAseq reaction using ERCC QC assay == Library Preparation (Unfragmented) == :'''RNA PREPARATION''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |2ng/ul, 200pg/ul, 20pg/ul UHRR||||||||||0.5ul |- |ERCC 1:10E4, 1:10E5, 1:10E6||||||||||0.2ul |- |STRT_V3_T30(10uM)||||||||||1.0ul |- |dNTP (10mM)||||||||||1ul |- |dH2O||||||||||1.8ul |- |Total||||||||||4.5ul |- |} :::''Incubate @ 65C 3 min'' :::''Transfer to ice'' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |5xRT Buffer||||||||||2ul |- |100mM DTT||||||||||0.17ul |- |STRT_V2_3 TSO (10uM)||||||||||1ul |- |Betaine||||||||||2ul |- |RNase Inhibitor (40U/ul)||||||||||0.1ul |- |Smarter MMLV (100U/ul)||||||||||0.23ul |- |Total||||||||||10ul |- |} :::''Incubate @ 42C 90 min'' :::''Incubate @ 70C 10 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 10ul dH2O, incubate 2min, transfer to new tube :'''PCR Amplification''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |cDNA||||||||||10ul |- |2x KAPA HiFi Hotstart ReadyMix||||||||||25ul |- |(10uM) STRT_PCR||||||||||1.0ul |- |dH2O||||||||||14ul |- |Total||||||||||50ul |- |} ::*98C 3min ::*12 cycles (1ng), 15 cycles (100pg) or 18 Cycles (10pg): :::*98C 15 sec :::*67C 20sec :::*72C 6min ::*72C 5min == Poly-A Unfragmented Library Preparation == :'''RNA PREPARATION''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |2ng/ul, 200pg/ul, 20pg/ul UHRR||||||||||0.5ul |- |ERCC 1:10E4, 1:10E5, 1:10E6||||||||||0.2ul |- |5x RT Buffer||||||||||0.5ul |- |1mM ATP||||||||||0.25ul |- |0.5U/ul PAP (1:10d in H2O)||||||||||0.25ul |- |dH20||||||||||0.8ul |- |Total||||||||||2.5ul |- |} :::''Incubate @ 37C 5 min'' :::''Transfer to Ice'' :'''REVERSE TRANSCRIPTION''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |PAP RNA ||||||||||2.5ul |- |10mM dNTP Mix||||||||||1ul |- |10uM STRT_V3_T30||||||||||1ul |- |dH2O||||||||||1.5ul |- |} :::''Incubate @ 65C 3 min'' :::''Transfer to Ice'' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |5xRT Buffer||||||||||1.5ul |- |100mM DTT||||||||||0.17ul |- |Betaine||||||||||2ul |- |RNase Inhibitor (40U/ul)||||||||||0.1ul |- |Smarter MMLV (200U/ul)||||||||||0.23ul |- |Total||||||||||10ul |- |} :::''Incubate @ 42C 90 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 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 Amplification''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |cDNA||||||||||10ul |- |2x KAPA HiFi Hotstart ReadyMix||||||||||25ul |- |STRT_PCR (10mM)||||||||||1.0ul |- |dH2O||||||||||14ul |- |Total||||||||||50ul |- |} ::*98C 3min ::*12 cycles (1ng), 15 cycles (100pg) or 18 Cycles (10pg): :::*98C 15 sec :::*67C 20sec :::*72C 6min ::*72C 5min == RNAseIII-Fragmented Library Preparation == :'''RNA PREPARATION''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |2ng/ul, 200pg/ul, 20pg/ul UHRR||||||||||0.5ul |- |ERCC 1:10E4, 1:10E5, 1:10E6||||||||||0.2ul |- |10x RNaseIII Buffer||||||||||0.2ul |- |1uM(1ng) or 0.1uM(100pg/10pg) T20V.id41 or STRT_V3_T30||||||||||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 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(1ng) or 0.1uM(100pg/10pg) T20V.id41 or STRT_V3_T30||||||||||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 to 3ul :'''TSO''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |Purified cDNA||||||||||3ul |- |5x RT buffer [MMLV]||||||||||2ul |- |10mM dNTP mix||||||||||1ul |- |100mM DTT||||||||||0.25ul |- |10uM STRT_V2_3 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'' :'''PhusionHF PCR (for T20V.ID)''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |TSO Reaction||||||||||11ul |- |2x Buffer||||||||||25ul |- |10uM P1-STRT||||||||||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 |- |} :'''KAPAHF PCR (STRT_V3_T30)''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |cDNA||||||||||11ul |- |2x KAPA HiFi Hotstart ReadyMix||||||||||25ul |- |STRT_PCR (10mM)||||||||||1.0ul |- |dH2O||||||||||13ul |- |Total||||||||||50ul |- |} ::*98C 3min ::*12 cycles (1ng), 15 cycles (100pg) or 18 Cycles (10pg): :::*98C 15 sec :::*67C 20sec :::*72C 30sec ::*72C 5min == RNAseIII-Fragmented Library Preparation (1-Step) == :'''RNA PREPARATION''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |2ng/ul, 200pg/ul, 20pg/ul UHRR||||||||||0.5ul |- |ERCC 1:10E4, 1:10E5, 1:10E6||||||||||0.2ul |- |10x RNaseIII Buffer||||||||||0.15ul |- |1uM(1ng) or 0.1uM(100pg/10pg) T20V.id41||||||||||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(1ng) or 0.1uM(100pg/10pg) T20V.id41||||||||||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 |- |} == Results of Amplification == *Run 5ul on gel [[File:09182013_PCR1_gel1.jpg]] == ERCC Quality Control Analysis == *Comparison of Pre-amp and post-amp plots: **ERCC-130-5'F/R **ERCC-130-midF/R **ERCC-130-3'F/R *Controls (carried through RT and Beads purification but no TSO/PCR - made up to 50ul): **Unfragmented STRT **Unfragmented Smart **PolyA Unfragmented STRT **Fragmented STRT **Fragmented T20V *Amplified 1ul control and 1ul PCR (total volume 25ul) using Sybr-Green: ::{| {{table}} |- |||||||||||<u>Volume</u> |- |Library||||||||||1ul |- |2xSybr Buffer||||||||||12.5ul |- |10uM F primer||||||||||0.5ul |- |10uM R Primer||||||||||0.5ul |- |dH2O||||||||||10.5ul |- |Total||||||||||25ul |- |} :::''95C 30sec'' :::''95C 10s -> 60C 20s -> 72C 30s'' => 40X :::''72C, 5min'' Loading Format for qPCR: {| {{table}} | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''Smart''' | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''Smart-PolyA''' | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''Smart-Frag''' | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''Smart-T20''' | align="center" style="background:#f0f0f0;"|'''''' |- | ||align="center" style="background:#f0f0f0;"|5'||align="center" style="background:#f0f0f0;"|mid||align="center" style="background:#f0f0f0;"|3'||align="center" style="background:#f0f0f0;"|5'||align="center" style="background:#f0f0f0;"|mid||align="center" style="background:#f0f0f0;"|3'||align="center" style="background:#f0f0f0;"|5'||align="center" style="background:#f0f0f0;"|mid||align="center" style="background:#f0f0f0;"|3'||align="center" style="background:#f0f0f0;"|5'||align="center" style="background:#f0f0f0;"|mid||align="center" style="background:#f0f0f0;"|3' |- | align="center" style="background:#f0f0f0;"|1ng PCR||Smart||Smart||Smart||Smart-PolyA||Smart-PolyA||Smart-PolyA||Smart-Frag||Smart-Frag||Smart-Frag||Smart-T20||Smart-T20||Smart-T20 |- | align="center" style="background:#f0f0f0;"|100pg PCR||Smart||Smart||Smart||Smart-PolyA||Smart-PolyA||Smart-PolyA||Smart-Frag||Smart-Frag||Smart-Frag||Smart-Frag||Smart-T20||Smart-T20 |- | align="center" style="background:#f0f0f0;"|10pg PCR||Smart||Smart||Smart||Smart-PolyA||Smart-PolyA||Smart-PolyA||Smart-Frag||Smart-Frag||Smart-Frag||Smart-Frag||Smart-T20||Smart-T20 |- | align="center" style="background:#f0f0f0;"|1ng RT||Smart||Smart||Smart||Smart-PolyA||Smart-PolyA||Smart-PolyA||Smart-Frag||Smart-Frag||Smart-Frag||Smart-Frag||Smart-T20||Smart-T20 |- | align="center" style="background:#f0f0f0;"|100pg RT||Smart||Smart||Smart||Smart-PolyA||Smart-PolyA||Smart-PolyA||Smart-Frag||Smart-Frag||Smart-Frag||Smart-Frag||Smart-T20||Smart-T20 |- | align="center" style="background:#f0f0f0;"|10pg RT||Smart||Smart||Smart||Smart-PolyA||Smart-PolyA||Smart-PolyA||Smart-Frag||Smart-Frag||Smart-Frag||Smart-Frag||Smart-T20||Smart-T20 | |- |} [[File:09182013_ERCC130_Smart.jpg]] Note: second chart compares 3' amplification for ERCC130 at different [RNA] [[File:09182013_ERCC130_Smart2.jpg]] [[File:09182013_ERCC130_Smart3.jpg]] == Final Library Preparation == *Beads purified (0.8x) *Re-amplified whole libraries 6x (1ng) or 7x (100pg, 10pg) *Beads purified (0.8x) *Ran 1ul on gel: [[File:09182013_PCR2_gel1.jpg]] Notes: *Library amplifications seemed low - might be an issue with one of the reagents e.g. MMLV. Therefore, need to repeat library production *Combined Smart/r03 (id9/12) (1ng seen above) for sequencing
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