title: GSM5259565: Polysome selection pool 1 selected cycle 3; Homo sapiens; OTHER
library_construction_protocol: Transfected RNA was isolated and fractionated by sucrose gradient as in the "polysome fraction" samples. Fractions 10-16 were pooled and RNA was purified by acidic phenol chloroform extraction followed by silica column purification (Zymo) 1.5 µg RNA in 5.5 µL was mixed with 0.5 µL 2uM RT_Nluc26_UMI12_Read1Partial (CCCTACACGACGCTCTTCCGATCTNNNNNNNNNNNNGGAACATCGTATGGGTAAACGGCCAT) and 0.5 µL 10 mM dNTPs each. The RNA samples were then denatured at 65°C for 5 min and chilled to 4°C. 3.5 µL reverse transcription mix was added to 10 µL total reaction volume: 2 µL 5x Superscript IV buffer, 0.5 µL 10 mM DTT, 0.5 µL Superase-In (ThermoFisher Scientific, AM2694), 0.5 µL Superscript IV (Thermo 18091050). The reaction was incubated at 55°C for 45 min and inactivated at 80°C for 10 min. For sequencing library preparation the RT reaction was PCR amplified under the 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 10 µM Read1, 1 µL 10 µM Read2Partial_HBB29 in 20 µL total reaction volume. Cycling conditions were as follows: 98°C for 60 sec, and 15 cycles of 98°C for 10 sec, 68°C for 10 sec, 72°C for 10 sec. Sequencing adaptors were added using the following conditions for final round PCR: 1 µL first round PCR reaction, 10 µL 2x Q5 Hot Start Master Mix, 0.2 µL 100x SYBR, 1 µL 10uM NEBNext Index Primer (NEB E7335, NEB E7500, NEB E7710, NEB E7730, NEB E6609), 1µL 10uM NEBNext Universal PCR Primer in 20 µL total volume. Cycling conditions are: 98°C for 60 sec, and 5 cycles of 98°C for 10 sec, 72°C for 10 sec. All barcoded samples were then pooled at equal volumes and purified with 1.1x SPRIselect beads Beckman Coulter B23317).