title: GSM4876246: 15.5K_CLASH_rep1; Homo sapiens; RNA-Seq
library_construction_protocol: Cell lysate was prepared and incubated with anti-FLAG. Afterwards, the RNA-protein complexes were immunoprecipitated with Dynabeads protein G and the RNA fraction was extracted using RNAzol reagent. HEK293T-FLAG-15.5K and HEK293T-FLAG-FBL cells grown on a 150 mm dish were rinsed with ice-cold PBS and crosslinked with 0.4 J/cm2 of 254 nm UV light. Crosslinked cells were harvested and snap frozen in liquid nitrogen and stored at -80°C. Frozen cell pellets were lysed directly in 6 volumes of ice-cold CLASH lysis buffer (50 mM Tris, pH 7.5, 100 mM NaCl, 0.1% SDS, 0.5% Sodium deoxycholate and 1% NP-40) supplemented with 1× protease inhibitor cocktail (Roche) on ice for 30 min. After sonication by using Bioruptor (Dieagenode) and then centrifugation at 15,000 g for 15 min at 4°C, the supernatant was treated with 60 U RQ1 DNase (Promega) at 37°C for 10 min, followed by adding 600 U S1 Nuclease for RNA digestion at 30°C for 15 min and stopped by addition of 80 μl 0.5 M EDTA. The lysate was precleared with Dynabeads Protein G (Invitrogen) at 4°C for 1 h and then subjected to FLAG antibody-bound beads for immunoprecipitation at 4°C overnight. After incubation, immunoprecipitaes were washed consecutively with once High-stringency buffer (20 mM Tris, pH 7.5, 120 mM NaCl, 5 mM EDTA, 25 mM KCl, 0.5% Sodium deoxycholate and 1% Triton X-100), once High-salt buffer (50 mM Tris, pH 7.5, 1 M NaCl, 0.1% SDS, 0.5% Sodium deoxycholate and 1% NP-40), once Low-salt buffer (50 mM Tris, pH 7.5, 5 mM EDTA) and twice NT2 buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 0.0005% NP-40 and 1 mM MgCl2). The immunoprecipitated material was digested with S1 Nuclease in final concentration of 4 U/µL at 30°C for 15 min and subsequently treated with T4 RNA Ligase 1 (NEB) for intramolecular ligation in an Eppendorf Thermomixer at 16 °C overnight, 15 s 1,400 r.p.m., 90 s rest in a 30 µL reaction. The beads were resuspended in 500 µL NT2 buffer and washed twice with High-salt buffer and twice with NT2 buffer. Then immunoprecipitaes were 3'-end ligated to IRdye-800CW-DBCO labeled preA-DNA adaptor with T4 RNA ligase 2 truncated KQ (NEB) in an Eppendorf Thermomixer at 16 °C overnight, 15 s 1,400 r.p.m., 90 s rest. (preA-DNA adaptor: /rApp/AGATCGGAAGAGCGGTTCAGAAAAAAAAAAAA/iAzideN/AAAAAAAAAAAA/3Bio/, /iAzideN/ site directly conjugated to IRdye-800CW-DBCO). The following day, beads were recovered and resuspended in 1× LDS sample buffer + reducing agent, and the RNA-protein complexes are run on NuPAGE™ 4-12% Bis-Tris Protein Gels and transferred to nitrocellulose membranes, and the region (30~120 nt of RNA for 15.5K and 50~130 nt of RNA for FBL) above the protein molecular weight is excised and Proteinase K (Thermo Fisher) treated to isolate RNA. The RNA was then reverse transcribed by using SuperScript IV reverse transcriptase (Invitrogen) and RT primer (ACACGACGCTCTTCCGATCT). After digestion of excessive RT primers, the cDNA was ligated to a 3' DNA adaptor (/5Phos/AGATCGGAAGAGCACACGTCTG/3SpC3/) using T4 RNA Ligase 1 (NEB). Next, the generated DNA was ampliﬁed by PCR that utilized 2 × Q5 PCR Master Mix (NEB) and corresponding illumina PCR primers. Ampliﬁed library was puriﬁed from 6% native acrylamide gel and sequenced on an Illumina HiSeq X ten platform.