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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_10pgUHRR_Jan13||UHRR/ERCC||10pg - RNase3 15s/Affymetrix PAP 4min||TSO.r06||T20VN_id1-10||N2.id15||totoRNAseq|||| |- | ||hNPC||SC - RNase3 15s/Affymetrix PAP 4min||TSO.r06||T20VN_id1-8,10-12||N2.id16||totoRNAseq|||| |- | ||hNPC||SC - RNase3 15s/Affymetrix PAP 4min||TSO.r06||T20VN_id13-23||N2.id17||totoRNAseq|||| |- | ||hNPC||10 cells - RNase3 15s/Affymetrix PAP 4min||TSO.r06||T20VN_id24||N2.id18||totoRNAseq|||| |- |} Note: hNPCs from [http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNA-Seq_Experiments:08302013] - excluding the 10pg control (id9) - combined for 10 cells per N2 index (~100pg total) Primer sequences [http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNAseq_Primer_List#totoRNA_5.27_primer_sequence] == Purpose == *Test optimized RNase III and PAP conditions on 10pg RNA based on [http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNA-Seq_Experiments:01082014 01082014] *Use ERCCs to determine level of transcript amplification between libraries *Combine libraries prior to TSO and amplification to reduce variability between samples and amplification cycle number *Compare with single cell sorted samples ([http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNA-Seq_Experiments:08302013]) == RNAseIII-Fragmented Library Preparation == :'''RNA''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |0.1ng/ul (for 10pg UHRR only)||||||||||0.1ul |- |1:10E6 (for 10pg UHRR only)||||||||||0.2ul |- |0.1uM T20VN||||||||||0.1ul |- |dH2O||||||||||0.6ul or 0.4ul for hNPCs |- |Total||||||||||1ul (0.5ul for hNPCs) |- |} :::''Incubate @ 72C 3 min'' :::''Incubate @ 37C 1 min'' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |10x RNaseIII buffer||||||||||0.2ul |- |RNase III||||||||||0.1ul |- |dH20||||||||||0.7ul |- |Total||||||||||2ul |- |} :::''Incubate @ 37C 15s-20s'' :'''3' Tailing''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |fRNA||||||||||2ul |- |5x RT buffer||||||||||0.5ul |- |1mM ATP||||||||||0.5ul |- |600U/ul Affymetrix PAP||||||||||0.2ul |- |40U/ul Rnase Inhibitor||||||||||0.2ul |- |dH2O||||||||||0.6ul |- |Total||||||||||4ul |- |} :::''Incubate @ 37C 4 min'' :::''Add 1ul of 0.1uM T20VN'' :'''Reverse Transcription''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |PAP RNA ||||||||||5ul |- |5xRT Buffer||||||||||1.5ul |- |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, combine wells ::#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 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||||||||||12x (100pg) |- |72C, 5min |- |} == Results of Amplification == *Run 5ul on gel: [[File:01132014_PCR1_Gel1.jpg|150px]] Results: 10 x 10pg UHRR and 10 x 10 hNPCs show similar amplification levels. 10 sorted hNPC cells (single well) shows less amplification - possibly due to the lower contribution of ERCCs and poorer lysis efficiency == Final Library Preparation == *Beads purified twice at (0.8x), re-suspended in 10ul *PCR on 0.5ul each library using ILMN_PCR_F and either ERCC130R227 or ERCC130R964. [[File:01132014_ERCC130_R227.jpg|400px]][[File:01132014_ERCC130_R964.jpg|400px]] *Results: ERCC130_R227 (5') PCR does not show much difference between samples - id18 had only a single well of ERCC and should be 10 fold less than the others... this does not make sense. ERCC_R964 (3'), however, does show the expected amplification distributions and indicates that the individual 10pg samples combined (100pg for 18 cycles) are similarly amplified as 1ng (for 15 cycles - exp 01082014). hNPC levels were also amplified similarly to the 10pg UHRR samples - sequencing would be needed to confirm and determine consistency between individual 10pg or hNPC single cell libraries.
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