GSM4159113: Day 24 - 1 TRAPseq; Homo sapiens; RNA-Seq
Polysomes were stabilized by adding 100 µg/ml cycloheximide to cell culture media for 10 minutes prior to homogenization. Cells were homogenized with a Teflon-glass homogenizer in ice-cold polysome extraction buffer (10 mM HEPES-KOH [pH 7.4], 150 mM KCl, 5 mM MgCl2, 0.5 mM DTT (MilliporeSigma, D9779-1G), 100 µg/ml cycloheximide (MilliporeSigma, C7698-1G), Superasin and RNasin RNase inhibitors (ThermoFisher, AM2694, PR-N2515)and complete-EDTAfree protease inhibitor (MilliporeSigma, 11836170001)). Following clearing by centrifugation, supernatants were incubated at 4˚C with end-over-end rotation for 16-18 hours with biotinylated Streptavidin T1 Dynabeads (ThermoFisher, 65601) previously conjugated with GFP antibodies (Sloan Kettering Institute Antibody Core, HtzGFP-19C8 and HtzGFP-19F7). The beads were collected on a magnetic rack, washed, and resuspended in lysis buffer with β-mercaptoethanol (Agilent, 400753) to extract bound RNA from polysomes. RNA was purified using the RNeasy micro kit (Qiagen, 74004). RNA quantity and quality was measured using an Agilent 2100 Bioanalyzer with the 6000 Pico Kit (Agilent, 5067-1513). 1 ng of total RNA was used to generate full length cDNA using Clontech's SMART-Seq v4 Ultra Low Input RNA Kit (Cat # 634888).  1 ng of cDNA was then used to prepare libraries using Illumina Nextera XT DNA sample preparation kit (Cat # FC-131-1024).   Libraries with unique barcodes were pooled at equal molar ratios and sequenced on Illumina NextSeq 500 sequencer to generate 75 bp single reads, following manufactures protocol (Cat# 15048776 Rev.E) cDNA amplification from mRNA followed by library prep
GEO Accession
GSM4159113