GSM8791325: Ribo-Seq, siIGF2BP3, ER stress, 2; Homo sapiens; OTHER
One ~80% confluent well of a 6-well plate per condition (three biological replicates per condition) was washed with ice-cold PBS (Sigma) supplemented with 100 mg/mL cycloheximide, lysed on a plate on ice with 350 μL of ice-cold lysis buffer (20 mM HEPES pH 7.3, 150 mM KCl, 5 mM MgCl2, 1% Triton X-100, 100 mg/mL cycloheximide, 1 mM DTT, 1x EDTA-free protease inhibitor cocktail), scraped, and transferred to 1.5 mL RNase-free tube. Cell lysates were passed three times through the 27G needle, incubated on ice for 10 min with intermittent vortexing, and clarified on a table-top centrifuge at maximum speed (20,000 g) for 20 min at 4°C. Total RNA concentration in the lysate was estimated using OD A260 measurement on Nanodrop. The lysate concentrations were brought to 240 ng/μL, 15 μL aliquots were taken for matching RNA-Seq. 200 μL of the lysate were supplemented with 5 mM CaCl2, 2 U/μL DNase I (NEB), 0.65 μg of micrococcal nuclease (Sigma, 10107921001), and 0.7 U of RNase I (Ambion) and incubated at room temperature for one hour on a rotator. The lysate was layered on 10%-50% sucrose gradient and centrifugated at 35,000 g for 3 hours at 4°C in a SW40Ti rotor. Polysome fractions were separated on a BioComp  fractionator and monosome fractions were collected. Ribo-Seq libraries were prepared as described in (McGlincy and Ingolia, 2017) with the following changes. rRNA was depleted from samples after adapter ligation using human riboPOOL probes (siTOOLs) according to the manufacturer's instructions, purified with Oligo Clean & Concentrator kit, and eluted in 12 μL of RNase-free water. Reverse transcription was done with SuperScript III (Invitrogen). Oligonucleotides used for Ribo-Seq are listed below. Preadenylated 5' adapter sequence is NNNNNNNNNNAGATCGGAAGAGCACACGTCTGAA. First five nucleotides of the adapter are UMI, while next five nuclotides are a sample index allowing multiplexing during library preparation. Samples barcodes are: ATCGT - siControl_DMSO, CGTAA - siIGF2BP2_DMSO, CTAGA - siIGF2BP3_DMSO, GATCA - siControl_TM, TAGAC - siIGF2BP2_TM, TCTAG - siIGF2BP3_TM. Reverse transcription primer sequence RNAGATCGGAAGAGCGTCGTGTAGGGAAAGAG/iSp18/GTGACTGGAGTTCAGACGTGTGCTC. Libraries were sequenced on a NovaSeq SP on SR100 (Illumina) at the Vienna BioCenter NGS facility with 60 million reads per sample on average.