GSM7036349: Chtop_mEx5_iCLIP_BR3: N2a Flp-In rtTA (Chtop_withoutEx5), Biological rep 3; Mus musculus; OTHER
iCLIP experiments were performed as previously described (Huppertz et al., 2014) with the addition of size-matched input (SMI) samples as negative controls (Van Nostrand et al., 2016). Cell pellets from crosslinked samples were lysed using ice cold lysis buffer (50 mM Tris–HCl, pH 7.4, 100 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate and 1 cOmplete™, Mini, EDTA-free Protease Inhibitor Cocktail tablet (Sigma Aldrich) per 10mL) at a 1:20 pellet weight to volume ratio (for 0.1g add 2.0 mL of lysis buffer) and sonicated. Lysates were divided into two 1 mL samples and treated with Turbo DNase (Thermo Fisher Scientiﬁc) and RNase I (Thermo Fisher Scientiﬁc) diluted 1/10 in PBS (HIGH RNase) or 1/50 in PBS (LOW RNase) for 5 minutes at 37°C to digest genomic DNA and partially digest RNA to short fragments of an optimal size range. RNA-protein complexes were purified using 100 µL of protein G Dynabeads (Thermo Fisher Scientiﬁc) and 2 μg of anti-Flag M2 (Sigma-Aldrich) antibody. 2% (20ul) of the total input lysate was retained for use as a SMI control. Following high salt washes, the immunoprecipitated RNA was 3′ end dephosphorylated and ligated to a pre-adenylated adaptor at the 3′ end overnight in a thermomixer (16 ºC), as described previously. The next day, 20% of the samples was 5′ end-labeled using radioactive ATP [γ-32P] and mixed with the unlabelled sample. The RNA-protein complexes from the immunoprecipitated and SMI samples were then separated on an SDS-PAGE gel and transferred to a nitrocellulose membrane overnight. RNA-protein complexes were isolated from the nitrocellulose membrane LOW RNase samples using the HIGH RNase samples as a reference (cut ~15-80 kDA above the protein length). RNA was recovered from the membrane by digesting the protein with proteinase k followed by phenol/chloroform extraction. SMI RNA samples were stored at -80 ºC until processing with 3'end adapter ligation and washes performed using MyOne Silane Dynabeads (Thermo Fisher Scientiﬁc) as per Van Nostrand et al., 2016. The RNA was next converted into cDNA using sample specific barcoded primers containing a BamH1 restriction site to allow for multiplexing and digestion (see below). The cDNA was size selected on a 6% TBE-Urea gel using two length cut-offs (Mid: 80–100 nt and High: 100-150 nt) and then circularized to add the 5'end adapter using CircLigaseTm II (Cambio). Circularized cDNA was digested using BamH1 and then PCR amplified using Phusion™ High-Fidelity DNA Polymerase (Thermo Fisher Scientiﬁc). PCR cycle numbers were optimized for both the Mid and High size selected fragments for both isoform samples (23-25 cycles for Mid and 27 cycles for High). The final PCR libraries were purified by agarose gel electrophoresis using gel extraction columns (QIAGEN), and eluted DNA mixed at a ratio of 1:1 from the Mid, and High fractions for sequencing. For Chtop +Ex5 isoform samples the barcode primers used were Rt6clip (BR1), Rt14clip (BR2) and Rt13clip (BR3) while for ∆Ex5 Chtop samples the primers used were Rt1clip (BR1), Rt10clip (BR2) and Rt9clip (BR3). For SMI sample libraries Rt16clip (BR1, GFP_input), Rt15clip (BR2_∆Ex5_input), and Rt2clip (BR3_+Ex5_input) were used. iCLIP-Seq