GSM8420859: SC_IN_rep1; Homo sapiens; RNA-Seq
MDA-MB-231 cells representing control and KD cells were cultured for 96 h in a puromycin medium (1 µg/ml). After that, the medium was changed with fresh, complete media. The following day, cells were treated with cycloheximide (CHX) (100 µg/ml) for 5 min and washed twice with cold 1X PBS containing CHX (100 µg/ml). Cells were further lysed in polysome lysis buffer (20mM Tris-HCl, pH 7.4, 5 mM MgCl2, 150 mM NaCl, 1% triton X-100) supplemented with 100 µg/ml cycloheximide, 1X complete protease inhibitors (complete EDTA-free, Roche), and 200U/ml of RiboLock RNase Inhibitor (ThemoScientific™). The lysates were tumbled for 10 minutes and then centrifuged at 10000 x g for 10 minutes at 4°C. Cleared lysates were normalised according to NanoDrop™ Lite Spectrophotometer (ThermoScientific™) measurements and layered on sucrose gradients (15-45% w/v) priorly prepared using Gradient Master (BioComp Instruments). The polyallomer tubes containing the sucrose gradients and the lysate were centrifuged at 39000xg for 2h at 4°C in an ultracentrifuge SW60 Ti rotor head (Beckmann). Fractions of 280 µl were collected using Gilson Fraction FC-203B Collector in tubes containing 300 µl Solution II (10 mM Tris-HCl pH 7.5; 350 mM NaCl; 10 mM EDTA; 1% SDS; 42% Urea) while continuously measuring the absorbance (A260) using the Triax™ flow cell (BioComp Instruments). For each sample, the absorbance of the lysate was measured with a NanoDrop spectrophotometer, and 10 µg of RNA was stored as input cytoplasmic RNA. The remaining lysate was treated with 4 U of DNase I (ThemoScientific™) and 800 U of RNase I (Ambion) for 45 min at R/T with gentle shaking. 800 U of RNasin ribonuclease inhibitor (Promega) was added to quench the reaction, and the samples were run on a 17.5–50% sucrose gradient to isolate monosomes. After separation through ultracentrifugation, the gradients were fractionated using an ISCO gradient fractionator (Brandel) and the fractions were collected in 300 µl of Solution II (see polysome profiling section). RNA from the monosome containing fraction and RNA Input samples were isolated using Phenol:Chloroform:Isoamyl alcohol (25:24:1). The RNA was quantified using a 2200 Agilent R6K ScreenTape station. Monosome-protected RNA fragments were dephosphorylated using T4 polynucleotide kinase (PNK) (Takara), and subsequently RNA was extracted using acid-phenol. Further, RNA was separated in a 15% polyacrylamide TBE-urea gel and footprint fragments 20-30 nucleotides (nt) in length were isolated from the gel. Sequencing libraries were prepared according to the protocol of the NEB NEXT Small RNA Library Prep Set for Illumina (Multiplex Compatible) E7330 kit. The cytoplasmic RNA (RNA Input) was sequenced using Illumina HiSeq V4 SR 50, while the footprint library used small RNA Seq-HiSeq V4 SR 50.