title: GSM8995259: deltaCTR mycLARP6-1; Homo sapiens; OTHER
library_name: GSM8995259
library_construction_protocol: For iCLIP, cells were UV-C crosslinked, lysed, and subjected to iCLIP as indicated in (https://doi.org/10.1101/2024.09.20.614075). Briefly, 20 million cells per condition were grown in 15 cm dishes and myc-LARP6 mutants were induced with 60 ng/mL doxycycline for 24 hours. Cells were crosslinked on ice at 150 mJ/cm2 at 254 nm in a Hoefer Scientific UV crosslinker to form covalent bonds between RBPs and RNAs that were in direct contact, then scraped in PBS and pelleted. Cell pellets were lysed in iCLIP lysis buffer (50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 1% Igepal CA-630, 0.1% SDS, 0.5% sodium deoxycholate, cOmplete protease inhibitor cocktail (Roche), 1 mM PMSF), cleared, and diluted to 1 mg/mL in 1 mL volume. Samples were then treated with 0.2 units/mL lysate RNase I (Thermo Scientific, catalogue number EN0602) and 2 μL Turbo DNase (Ambion) for 3 minutes at 37°C to partially digest the RNA. 50 μL Dynabeads Protein G (Invitrogen) per sample were first conjugated with 4 μg (10 μL) mouse anti-myc-tag antibody (Cell Signaling, 9B11) for one hour at RT in a rotating wheel, then added to the samples to perform the immunoprecipitation (IP) for one hour at 4°C in a rotating wheel. After the IP, samples were stringently washed twice with high-salt wash buffer (50 mM Tris-HCl, pH 7.4, 1 M NaCl, 1 mM EDTA, 1% Igepal CA-630, 0.1% SDS, 0.5% sodium deoxycholate), then once with PNK wash buffer (20 mM Tris-HCl, pH 7.4, 10 mM MgCl2, 0.2% Tween-20). For Input RNA-seq, cells grown under the same conditions as the ones used in the iCLIP experiment was subjected to RNA extraction with TRIzol. The purified RNAs were quantified with the Qubit RNA High-sensitivity assay, and the quality was validated with the Agilent High Sensitivity RNA ScreenTape assay, with all samples having a RIN ≥ 9.3. The whole transcriptome RNAseq library preparation and sequencing was outsourced to Novogene. For iCLIP, The RNA fragments were dephosphorylated on the beads at the 3' end, followed by 3' end ligation of different barcoded adapters, to allow for downstream multiplexing of each replicate into one sample. The samples were then eluted from the beads, run on an SDS-PAGE gel, followed by isolation of the RNA fragments and subsequent library preparation as described previously. The amplified libraries were quantified and validated with the Qubit dsDNA High-sensitivity and Agilent D1000 high sensitivity ScreenTape assays, pooled in an equimolar ratio, and deep sequenced on an Illumina NextSeq 500 instrument, using a single-end 100 nt workflow. Sequencing was performed at the Barts and The London Genome Centre. For Input RNA-seq, rRNA depletion whole transcriptome sequencing using two protocols (short and long RNAs) was performed by Novogene (https://www.novogene.com/eu-en/services/research-services/transcriptome-sequencing/whole-transcriptome-sequencing/)