title: GSM5259551: RNA in-solution timepoint 2; Homo sapiens; OTHER
library_construction_protocol: RNA was purified using Ampure beads + 40% polyethylene glycol 8000 (7:3) Up to 250 ng RNA in 2.75 µL was mixed with 0.25 µL 2 µM RT_Const2_N12_Read1Partial (TCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNNNNNNNtcaccacggcgtgagatcagcg) and 0.25 µL 10mM dNTPs each. The RNA samples were then denatured at 65°C for 5 min and chilled to 4°C. 1.75 µL reverse transcription mix was added to 5 µL total reaction volume: 1 µL 5x Superscript IV buffer, 0.25 µL 10mM DTT, 0.25 µL Superase-In (ThermoFisher Scientific, AM2694), 0.25 µL Superscript IV (Thermo 18091050). The reaction was incubated at 55°C for 45 min and inactivated at 80°C for 10 min. First round PCR was performed under following conditions: 1 µL RT reaction, 10 µL 2x Q5 Hot Start Master Mix (NEB M0494S), 0.2 µL 100x SYBR (Thermo S7563), 1 µL 10uM Read1Partial_F (TCTTTCCCTACACGACGCTCTTCCGATCT), 1 µL 10 uM 50:50 Hbb_Fwd:Nluc_Fwd mix in 20 µL total volume. Cycling conditions were: 98°C for 60 sec, and 15 cycles of 98°C for 10 sec, 68°C for 10 sec and 72°C. Second round PCR was performed under the following conditions: 1 µL first round PCR, 10 µL 2x Q5 Hot Start Master Mix, 0.2 µL 100x SYBR, 1 µL 10 uM Read1Partial_F, 1 µL 10 uM Read2Partial_Const1_R (GGAGTTCAGACGTGTGCTCTTCCGATCTttctagagcggccgcttcgagc) in 20 µL total volume. Cycling conditions were: 98°C for 60 sec, and 5 cycles of 98°C for 10 sec, 72°C for 5 sec. Sequencing adaptors were added using the following conditions for final round PCR: 1 µL second round PCR, 10 µL 2x Q5 Hot Start Master Mix, 0.2 µL 100x SYBR, 1 µL 10 µM NEBNext Index Primer (NEB E7335, NEB E7500, NEB E7710, NEB E7730, NEB E6609), 1 µL 10 µM NEBNext Universal PCR Primer in 20 µL total volume. Cycling conditions were: 98°C for 60 sec, and 5 cycles of 98°C for 10 sec, 72°C for 5 sec. All barcoded samples were then pooled at equal volumes and purified with 1.1x SPRIselect beads (Beckman Coulter B23317).