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== Final Library Preparation == ''cDNA Quantification'' :'''Quantification of 1ul out of 10ul Beads purified libraries using Qubit:''' {| {{table}} | align="center" style="background:#f0f0f0;"|'''Sample''' | align="center" style="background:#f0f0f0;"|'''Condition''' | align="center" style="background:#f0f0f0;"|'''[cDNA] ng/ul''' | align="center" style="background:#f0f0f0;"|'''Final Library''' | align="center" style="background:#f0f0f0;"|'''Volume to Combine''' | align="center" style="background:#f0f0f0;"|'''N2 Barcode''' | align="center" style="background:#f0f0f0;"|'''NexteraXT''' | align="center" style="background:#f0f0f0;"|'''Volume for 0.1-0.2ng/ul''' | align="center" style="background:#f0f0f0;"|'''Volume dH2O''' | align="center" style="background:#f0f0f0;"|'''Nextera Barcode''' | align="center" style="background:#f0f0f0;"|'''Sten Tagmentation''' | align="center" style="background:#f0f0f0;"|'''Volume for 1ng/ul''' | align="center" style="background:#f0f0f0;"|'''Volume dH20''' | align="center" style="background:#f0f0f0;"|'''Sten Barcode''' |- | 1||TotoRNAseq Mg 1ng||8.95||Yes||2||1||N/A||||||||N/A|||||| |- | 2||TotoRNAseq Mg 1ng||8.84||Yes||2.02||2||N/A||||||||N/A|||||| |- | 3||TotoRNAseq Mg 100pg||8.57||Yes||2.09||3||N/A||||||||N/A|||||| |- | 4||TotoRNAseq Mg 100pg||10||Yes||1.79||4||N/A||||||||N/A|||||| |- | 5||TotoRNAseq Mg 10x10pg||15.2||Yes||N/A||5||N/A||||||||N/A|||||| |- | 6||TotoRNAseq Mg NTC||4.04||Yes||2||6||N/A||||||||N/A|||||| |- | 7||TotoRNAseq RIII 1ng||3.13||Yes||1||7||N/A||||||||N/A|||||| |- | 8||TotoRNAseq RIII 1ng||2.96||Yes||1.06||8||N/A||||||||N/A|||||| |- | 9||TotoRNAseq RIII 100pg||1.38||Yes||2.27||9||N/A||||||||N/A|||||| |- | 10||TotoRNAseq RIII 100pg||0.977||Yes||3.20||10||N/A||||||||N/A|||||| |- | 11||TotoRNAseq RIII 10x10pg||1.39||Yes||N/A||11||N/A||||||||N/A|||||| |- | 12||TotoRNAseq RIII NTC||-||Yes||2||12||N/A||||||||N/A|||||| |- | 13||TotoRNAseq RIII StenTSO 1ng||1.21||Yes||1||13||N/A||||||||N/A|||||| |- | 14||TotoRNAseq RIII StenTSO 1ng||1.54||Yes||0.785714286||14||N/A||||||||N/A|||||| |- | 15||TotoRNAseq RIII StenTSO 100pg||0.948||Yes||1.276371308||15||N/A||||||||N/A|||||| |- | 16||TotoRNAseq RIII StenTSO 100pg||0.844||Yes||1.433649289||16||N/A||||||||N/A|||||| |- | 17||TotoRNAseq RIII StenTSO 10x10pg||0.19||Yes||6.368421053||17||N/A||||||||N/A|||||| |- | 18||TotoRNAseq RIII StenTSO NTC||-||Yes||1||18||N/A||||||||N/A|||||| |- | 19||STRT RIII 1ng||3.54||No||||||N/A||||||||Yes||1.694915254||4.305084746||C1-TN5-1 |- | 20||STRT RIII 1ng||3.35||No||||||N/A||||||||Yes||1.791044776||4.208955224||C1-TN5-2 |- | 21||STRT RIII 100pg||2.78||No||||||N/A||||||||Yes||2.158273381||3.841726619||C1-TN5-3 |- | 22||STRT RIII 100pg||2.99||No||||||N/A||||||||Yes||2.006688963||3.993311037||C1-TN5-4 |- | 23||STRT RIII 10pg||2.42||No||||||N/A||||||||Yes||2.479338843||3.520661157||C1-TN5-5 |- | 24||STRT RIII 10pg||2.22||No||||||N/A||||||||Yes||2.702702703||3.297297297||C1-TN5-6 |- | 25||STRT RIII 10pg||2.86||No||||||N/A||||||||Yes||2.097902098||3.902097902||C1-TN5-7 |- | 26||STRT RIII NTC||0.696||No||||||N/A||||||||Yes||6||0||C1-TN5-8 |- | 27||Smart2 RIII 1ng||1.48||No||||||Yes||0.68||4.32||S501/N701||N/A|||||| |- | 28||Smart2 RIII 1ng||1.47||No||||||Yes||0.68||4.32||S502/N702||N/A|||||| |- | 29||Smart2 RIII 100pg||0.52||No||||||Yes||1.92||3.08||S503/N703||N/A|||||| |- | 30||Smart2 RIII 100pg||0.49||No||||||Yes||2.04||2.96||S504/N704||N/A|||||| |- | 31||Smart2 RIII 10pg||0.13||No||||||Yes||3.85||1.15||S505/N705||N/A|||||| |- | 32||Smart2 RIII 10pg||0.12||No||||||Yes||4.17||0.83||S506/N706||N/A|||||| |- | 33||Smart2 RIII 10pg||-||No||||||Yes||5.00||0.00||S507/N707||N/A|||||| |- | 34||Smart2 RIII NTC||-||No||||||Yes||5.00||0.00||S508/N708||N/A|||||| |- | 35||Quartz RIII 1ng||13.8||No||||||Yes||0.58||39.42||S501/N709||N/A|||||| |- | 36||Quartz RIII 1ng||10.4||No||||||Yes||0.77||39.23||S502/N710||N/A|||||| |- | 37||Quartz RIII 100pg||3.49||No||||||Yes||0.86||14.14||S503/N711||N/A|||||| |- | 38||Quartz RIII 100pg||3.73||No||||||Yes||0.80||14.20||S504/N712||N/A|||||| |- | 39||Quartz RIII 10pg||1.95||No||||||Yes||1.54||13.46||S505/N701||N/A|||||| |- | 40||Quartz RIII 10pg||3.63||No||||||Yes||0.83||14.17||S506/N702||N/A|||||| |- | 41||Quartz RIII 10pg||4.93||No||||||Yes||0.61||14.39||S507/N703||N/A|||||| |- | 42||Quartz RIII NTC||1.83||No||||||Yes||1.64||13.36||S508/N704||N/A|||||| |} ''Nextera Tagmentation'' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |Tagment DNA Buffer||||||||||5ul |- |Amplification Tagment Mix||||||||||2.5ul |- |Diluted Sample||||||||||2.5ul |- |Total||||||||||10ul |- |} ::*Vortex and spin ::*Incubate 55C 5 min ::*Hold 10C ::*Once sample reaches 10C add 2.5ul of NT buffer in block ::*Vortex and spin ''Nextera Amplification'' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |Tagmented Sample||||||||||12.5ul |- |Nextera PCR Mix||||||||||7.5ul |- |S5xx Primer||||||||||2.5ul |- |S7xx Primer||||||||||2.5ul |- |Total||||||||||20ul |- |} ::''Mix and centrifuge'' ::{| {{table}} |- |72C 3 min|||||||||| |- |95C 30s|||||||||| |- |95C 10s -> 55C 30s -> 72C 60s||||||||||12x |- |72C, 5min |- |} ::*Run 2.5ul on gel: [[File:03052014_PCR2_Gel1_Nextera.jpg|500px]] ::*Combined 5ul of each for Smart2 ::*Combined 5ul each of 1ng/100pg/NTC and 7.5ul each of 10pg for Quartz-seq ::*Beads purified twice (0.8x volume) and resuspended in 10ul ''Sten Tagmentation'' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |5x Tagmentation Buffer||||||||||5ul |- |Transposome stock (Epicenter)||||||||||6ul |- |Sample||||||||||3ul |- |dH2O||||||||||11ul |- |Total||||||||||25ul |- |} ::*Incubate @55C for 8 min ::*Cool on ice ::*Wash 100ul Dynabeads MyOne Streptavidin C1 Beads with 2xBWT and resuspended in 2ml 2xBWT ::*Add 20ul beads to each well and incubate 5min ::*Combine beads, immobilize and discard supernatant ::*Wash beads with 100ul TNT ::*Wash beads with 100ul Qiagen Qiaquick PB ::*Wash beads twice with 100ul TNT ::*Resuspend beads in 100ul restriction mix: :::*10ul NEBuffer 4 :::*2ul PvuI-HF (0.4U/ul) :::*88ul dH2O ::*Incubate at 37C for 1 hour ::*Wash beads 3x with TNT ::*Elute in 10ul 100mM NaOH, incubate 5 min then remove to new tube ::*Neutralize by adding: :::*10ul 100mM HCl :::*5ul Neutralization buffer ::*Check library size by gel (ran 5ul on UREA gel): [[File:03052014_PCR2_Gel1_STRT.jpg|100px]] ::Ran Sybr PCR using C1_P1_PCR_2/C1_TN5_U for 12 cycles (25ul total) and ran 5ul on gel: [[File:03052014_PCR2_Gel2_STRT.jpg|100px]] ::[http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:Data/2014-5-12 Final Libraries Ran on Gel]
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