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==Making ds-cDNA for Hybrids/BJ== * Total RNA Samples: o A: Hybrid1-ES: 750ng/ul o B: Hybrid1-ES: 1.9ug/ul o C: BJ: 520ng/ul Clean up and concentrate all RNAs with Zymo DNA-Free RNA kit A B C RNA 6ul 2.5ul 10ul 10X DNase I buffer 5ul 5ul 5ul RNase-Free DNase I 2.5ul 2.5ul 2.5ul H2O 36.5ul 40ul 32.5ul 37C 10min * Add 4 volumes of RNA binding buffer; * Transfer to Zymo-spin columns in collection tubes, spin @15k rpm for 30s; * Add 200ul RNA Wash buffer, spin @15k rpm for 30s; * Add 10ul DNase/RNase-free water, spin @15k rpm for 30s; Reverse transcription A B C RNA 8ul 8ul 8ul 10mM dNTP 1ul 1ul 1ul 50uM dT12-18 1ul 1ul 1ul 65C 5min -> chill on ice for 1min Add 10X RT buffer 2ul 2ul 2ul 25mM MgCl2 4ul 4ul 4ul 0.1M DTT 2ul 2ul 2ul RNaseOUT 1ul 1ul 1ul Superscript III 1ul 1ul 1ul Incubate all tube at 50C for 50min. 2nd strand synthesis Cleanup the first-strand reactions with qiaquick columns. To the 30ul reactions, add 10ul NEBuffer 2 5ul 10mM dNTP 2.5ul DNA Pol I(10U/ul) 1ul RNaseH 1ul H2O 10.5ul Incubate at 16C for 2h, purified with Qiaquick columns. Yield (use DNA's absorbance): RNA A: Hybrid1-ES(750ng/ul): 22.9ng/ul x 30ul 18.1ng/ul B: Hybrid1-ES(1.9ug/ul): 80.2ng/ul x 30ul 63.4ng/ul C: BJ(520ng/ul): 94.4ng/ul x 30ul 72.3ng/ul
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