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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/ERCC||1ng ||STRT_v2_3||STRT_V3_T30||||Smart-Seq2|||| |- | BL_||UHRR/ERCC||1ng ||STRT_v2_3||STRT_V3_T30||||Smart-Seq2|||| |- | BL_||UHRR/ERCC||1ng - Poly(A)||STRT_v2_3||STRT_V3_T30||||Smart-Seq2|||| |- | BL_||UHRR/ERCC||1ng - Poly(A)||STRT_v2_3||STRT_V3_T30||||Smart-Seq2|||| |- | BL_||UHRR/ERCC||1ng - Poly(A) with dATP||STRT_v2_3||STRT_V3_T30||||Smart-Seq2|||| |- | BL_||UHRR/ERCC||1ng - Poly(A) with dATP||STRT_v2_3||STRT_V3_T30||||Smart-Seq2|||| |- |} == Purpose == *To test the effectiveness of Smart-Seq2 protocol for partially degraded samples and the potential rescuing effect of PAP == Smart-seq 2 Protocol == :'''RNA''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |UHRR 2ng/ul ||||||||||0.5ul |- |ERCC 1:10E4||||||||||0.2ul |- |dH2O||||||||||0.3ul |- |10uM T30||||||||||1ul |- |dNTP (10mM)||||||||||1ul |- |Total||||||||||3ul |- |} :::''Incubate @ 72C 3 min'' :::''Transfer to ice'' :'''Reverse Transcription''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |5xRT Buffer||||||||||2ul |- |100mM DTT||||||||||0.25ul |- |100mM MgCl2||||||||||0.6ul |- |Betaine (5M)||||||||||2ul |- |TSO||||||||||1ul |- |dH20||||||||||0.4ul |- |RNase Inhibitor (40U/ul)||||||||||0.25ul |- |Smarter MMLV (200U/ul)||||||||||0.5ul |- |Total||||||||||10ul |- |} :::''Incubate @ 42C 90 min'' :::''Incubate @ 70C 10 min'' :'''Beads Purification''' ::#Add 25ul beads per well ::#Allow to bind 15 min ::#Remove supernatant and wash 2x with 1ml 80% EtOH ::#Air Dry until cracking first occurs ::#Resuspend beads in PCR reaction: :'''PCR (Advantage)''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |10x Advantage 2 PCR Buffer||||||||||5ul |- |dNTP Mix (10mM)||||||||||2ul |- |STRT Primer (10uM)||||||||||2ul |- |50X Advantage 2 Polymerase Mix||||||||||2ul |- |H2O||||||||||39ul |- |Total||||||||||50ul |- |} ::{| {{table}} |- |95C 1 min|||||||||| |- |95C 15s -> 65C 30s -> 68C 6min||||||||||12x |- |72C, 10min |- |} == Smart-seq 2 Protocol with Poly(A) Tailing == :'''RNA''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |UHRR 2ng/ul ||||||||||0.5ul |- |ERCC 1:10E4||||||||||0.2ul |- |5x ePAP Buffer||||||||||0.6ul |- |dH2O||||||||||0.9ul |- |Total||||||||||2.2ul |- |} :::''Incubate @ 72C 3 min'' :::''Transfer to ice'' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |25mM MnCl2||||||||||0.3ul |- |1mM ATP or 1:1 ATP/dATP||||||||||0.25ul |- |ePAP 2u/ul||||||||||0.25ul |- |Total||||||||||3ul |- |} :::''Incubate @ 37C 10 min'' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |10uM T30||||||||||1ul |- |dNTP (10mM)||||||||||1ul |- |Total||||||||||5ul |- |} :'''Reverse Transcription''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |5xRT Buffer||||||||||2ul |- |100mM DTT||||||||||0.25ul |- |100mM MgCl2||||||||||0.3ul |- |Betaine (5M)||||||||||2ul |- |RNase Inhibitor (40U/ul)||||||||||0.25ul |- |Smarter MMLV (200U/ul)||||||||||0.5ul |- |Total||||||||||10.3ul |- |} :::''Incubate @ 42C 90 min'' :'''Beads Purification''' ::#Add 25ul beads per well ::#Allow to bind 15 min ::#Remove supernatant and wash 2x with 1ml 80% EtOH ::#Air Dry until cracking first occurs ::#Resuspend beads in TSO reaction: :'''TSO''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |dH2O||||||||||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 (Advantage)''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |10x Advantage 2 PCR Buffer||||||||||5ul |- |dNTP Mix (10mM)||||||||||2ul |- |STRT Primer (10uM)||||||||||2ul |- |50X Advantage 2 Polymerase Mix||||||||||2ul |- |H2O||||||||||29ul |- |Total||||||||||50ul |- |} ::{| {{table}} |- |95C 1 min|||||||||| |- |95C 15s -> 65C 30s -> 68C 6min||||||||||12x |- |72C, 10min |- |} == Results of Amplification == *Run 5ul on gel: [[File:11072013_PCR1_gel1.jpg]] *Results: Can see that the overall efficiency is abnormally low. We have been having a problem with this and the Smart-Seq protocol lately for some unknown reason. Poly(A) tailing clearly increases the yields of cDNA. == Final Library Preparation ==
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