title: GSM5259525: Polysome fraction 7; Homo sapiens; OTHER
library_construction_protocol: Cell culture media was replaced with cycloheximide (MilliporeSigma, C7698-1G) containing media at 100ug/mL. After 2 minutes, cells were washed, trypsinized and harvested using PBS, trypsin, and culture media containing 100 g/mL cycloheximide. ~10x106 cells were resuspended in 400 µL of following lysis buffer on ice for 30 min, vortexing every 10 min: 25 mM Tris-HCl pH 7.5, 150 mM NaCl, 15 mM MgCl2, 1 mM DTT, 8% glycerol, 1% Triton X-100, 100 µg/mL cycloheximide, 0.2 U/µL Superase-In RNase inhibitor (ThermoFisher Scientific, AM2694), 1x Halt protease inhibitor cocktail (ThermoFisher Scientific, 78430), 0.02 U/µL TURBO DNase (ThermoFisher Scientific, AM2238). After lysis, nuclei were removed by two step centrifuging, first at 1300 g for 5min and second at 10000 g for 5min, taking the supernatants from each. 25%-50% sucrose gradient was prepared in 13.2mL ultracentrifuge tubes (Beckman Coulter, 331372) using Biocomp Gradient Master with the following recipe: 25 or 50% sucrose (w/v), 25 mM Tris-HCl pH 7.5, 150 mM NaCl, 15 mM MgCl2 , 1 mM DTT, 100 µg/mL cycloheximide. The lysate was layered onto the sucrose gradient and ultracentrifuged on Beckman Coulter SW-41Ti rotor at 40000rpm for 150min at 4°C. The gradient was density fractionated using Brandel BR-188 into 16x750 µL fractions, and in vitro transcribed spike-in RNA mix (120002B1, 120010B1, 220023B1, 310333T3; 1000, 100, 10, 1-fold dilutions respectively) were added to each fraction. 700 µL of each fraction was mixed with 100µL 10% SDS, 200µL 1.5M sodium acetate, and 900µL acid phenol-chloroform, pH 4.5 (ThermoFisher Scientific, AM9720), heated at 65°C for 5min, and centrifuged at 20000g for 15min at 4°C for phase separation. 600µL aqueous phase was mixed with 600 µL 100% ethanol and RNA was purified on silica columns (Zymo, R1013). 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).