GSM4697860: RPF_4sU20_exp2; Mus musculus; RNA-Seq
Cells were washed once in ice-cold 1 x PBS supplemented with 100 µg/ml cycloheximide (Roth, 8682.3), and harvested by scraping in polysome lysis buffer (20 mM Tris-HCl pH 7.4, 10 mM MgCl2, 200 mM KCl2, 1% NP-40, 100 µg/ml cycloheximide, 2 mM DTT, 1 tablet EDTA-free Roche cOmplete Mini Protease Inhibitor per 10 ml). Lysates were rotated end-over-end for 10 min at 4°C and cleared by centrifugation at 9,300 g for 10 min at 4°C. About 10% of the lysates were saved as input samples. The lysates were subsequently digested with RNase I (240 U per A260; Ambion AM2294) for 5 min at 4°C. Samples were then subjected to 17.5–50% sucrose density gradient centrifugation (for 1 h 45 min at 40,000 rpm and 4°C in a SW60 rotor) and fractions of 250 – 300 µl were collected with a Teledyne Isco Foxy Jr. fractionator directly into 300 µl urea buffer (10 mM Tris-HCl ph 7.5, 350 mM NaCl, 10 mM EDTA, 1% SDS, 7 M urea). RNA was purified from the cytoplasmic lysate (input) or from the monosomal fractions (ribosome footprint) using phenol:chloroform:isoamylalcohol (25:24:1, AppliChem A0944) by phase separation and precipitation with GlycoBlue (Ambion AM9515) in 50 – 60% isopropanol. Both input and ribosome protected fragments were depleted of ribosomal RNA (rRNA) with the Ribo-Zero Gold Kit (Illumina MRZG126). Input RNA was randomly fragmented by alkaline hydrolysis at pH 10.0 for 12 min at 95°C. Per sample, 0.5 ng spike-in RNA (5'-ATCTACGCGCGA-4sU-AAGGCTAAGCTAGGCACC-3') was added. Conversion of 4sU to cytidine was induced by treatment with 10 mM NaIO4 in 600 mM NH4Cl pH 8.5 for 15 min at 40°C in a thermomixer. After the conversion reaction, RNA was recovered with G-25 mini Quick Spin Oligo Columns (Roche). Fragmented RNA and ribosome protected fragments were size-selected (25 – 35 nt) on a 15% polyacrylamide Tris-borate-EDTA-urea gel after staining with SybrGold. RNA was eluted from the gel slices by rotating at 4°C overnight in 300 mM NaCl plus RNase OUT (Invitrogen). The gel matrix was removed by centrifugation in a 0.45 µM NanoSep MF tube (PALL). After precipitation with isopropanol and GlycoBlue, end-repair was performed with 10 U T4 PNK (NEB M0201S), 40 U RNase OUT and 1 mM ATP in T4 PNK reaction buffer for 1 h at 37°C. After end-repair with T4 PNK, libraries were prepared with the NEXTflex Small RNA-Seq Kit v3 according to the manufacturer's manual, using 5 ng RNA for experiment 1 and 8 ng for experiment 2. In order to determine the number of PCR cycles required to obtain a ~ 20 nM library, 1 µl of cDNA per sample was diluted 1:8 and used for a SybrGreen qPCR (forward primer: 5'-GTTCAGAGTTCTACAGTCCGA-3', reverse primer: 5'-CCTTGGCACCCGAGAATTCCA-3', SybrGreen master mix: applied biosystems A25742) on a QuantStudio Real-Time PCR System. For library preparation, three cycles less than the highest determined cycle of threshold were used (12 cycles for experiment 1 and 11 cycles for experiment 2).