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== Quartz-Seq Library Preparation == '''RT Primer Removal''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |Purified cDNA||||||||||3ul |- |10x PCR Buffer||||||||||0.4ul |- |10x Exo I buffer||||||||||0.2ul |- |100mM DTT||||||||||0.2ul |- |Exonuclease I||||||||||0.6ul |- |dH2O||||||||||1.6ul |- |Total||||||||||6ul |- |} ''Note: can add directly to beads and transfer to new tube'' :::''Incubate @37C 30min'' :::''Incubate @80C 20min'' :::''Incubate @4C'' '''Poly-A Tailing''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |ExoI RXN||||||||||6ul |- |10x PCR Buffer||||||||||0.5ul |- |100mM dATP||||||||||0.15ul |- |RNaseH||||||||||0.12ul |- |TdT enzyme||||||||||0.42ul |- |dH2O||||||||||3.81ul |- |Total||||||||||11ul |- |} :::''Incubate @37C 50 sec'' :::''Incubate @65C 10min'' :::''Incubate @4C'' '''Second Strand Synthesis''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |Poly-A Tailed Sample||||||||||11ul |- |2x Terra Direct PCR Buffer||||||||||25ul |- |10uM Tagging Primer||||||||||0.32ul |- |Terra Polymerase||||||||||2ul |- |dH2O||||||||||18.68ul |- |Total||||||||||57ul |- |} :::''Incubate @98C 2min 10 sec'' :::''Incubate @40C 1min'' :::''Incubate @68C 5min'' :::''Transfer to ice'' '''PCR Amplification''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |2nd Strand Sample||||||||||57ul |- |2x Terra Direct PCR Buffer||||||||||25ul |- |100uM PCR Primer||||||||||1ul |- |dH2O||||||||||24ul |- |Total||||||||||107ul |- |} ::{| {{table}} |- |68C 1 sec|||||||||| |- |98C 10s -> 65C 15s -> 68C 5min||||||||||15x (1ng); 18x (100pg); 21x (10pg) |- |68C, 5min |- |} Ran 5ul onto gel: [[File:03052014_PCR1_Gel1_Quartz.jpg|500px]] Results: Mg protocol also is problematic for Quartz-seq. I'm not sure why this is the case since no RNA adaptor primer was used... Looks like the RNaseIII protocol did work down to the 10pg level, though quantification is needed to determine whether there is enough yield for Nextera library preparation. Note that while the yields look lower, less sample was run on the gel compared to totoRNAseq (5ul of 107ul vs 5ul of 50ul).
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