GSM9017060: TTchemDRBseq, CFAP20-KO + TY-CFAP20-R100C, 60min, rep2; Homo sapiens; OTHER
Immediately after 4SU labeling, cells were washed with PBS and lysed in TRIzol reagent (Invitrogen). Total RNA was isolated by TRIzol/chloroform isolation and ethanol precipitation. 110 μg RNA (in a total volume of 116 μl) was fragmented by adding 4 μl of 5 M NaOH and left on ice for 30 min. Fragmentation was stopped by the addition of 80 μl of 1 M Tris pH 7 and cleaned up twice using the Micro Bio-Spin P-30 Gel Columns (BioRad, 7326223) according to the manufacturer's instructions. Biotinylation of 4SU-residues was performed in a total volume of 250 μl, containing 10 mM Tris-HCl pH 7.4, 1 mM EDTA and 5 μg of MTSEA biotin-XX linker (Biotium, BT90066) for 30 min at room temperature in the dark. RNA was then purified by phenol-chloroform extraction, denatured by 10 min incubation at 65 °C and added to 150 μl μMACS Streptavidin MicroBeads (Milentyl, 130-074-101). RNA was incubated with beads for 15 min at room temperature and beads were applied to a μColumn in the magnetic field of a μMACS magnetic separator. The beads were washed tree times with 55°C pull-down wash buffer (100 mM Tris-HCl, pH 7.4, 10 mM EDTA, 1 M NaCl and 0.1% Tween-20). Biotinylated RNA was eluted twice by addition of 100 mM DTT and cleaned up using the RNeasy MinElute kit (Qiagen, 74204) using 1,050 μl ethanol (≥99%) per 200 μl reaction after addition of 700 μl of RLT buffer to precipitate RNA of less than 200 nucleotides. For WT and CFAP-KO cells, purified 4SU-labelled RNA was then depleted of rRNAs using the QIAseq FastSelect -rRNA depletion kit (Qiagen), followed by library preparation using the TruSeq Stranded Total RNA kit (Illumina, 20020596). The libraries were amplified according to the manufacturer's instructions, pooled and paired-end sequenced on a DNBSEQ-G400 (BGI) system. For CFAP20-KO cells expressing GFP-CFAP20(WT) or GFP-CFAP20(R100C), no ribodepletion was performed.