GSM4512279
GSM4512279: HEK293T_IP_CLK3_rep1; Homo sapiens; OTHER
Cell lysates were prepared by sonication in 1 ml iCLIP lysis buffer, treated with RNase I (40 U, LifeTech). Enhanced crosslinking and immunoprecipitation (eCLIP) was performed on HEK293T cells, and immunoprecipitated overnight at 4 °C with 10 µg affinity-purified antibody pre-coupled to Dynabeads sheep M-280 anti-IgG beads (LifeTech). Prior to IP, a 20 µl aliquot of extract was removed and stored at 4 °C for preparation of the size-matched input (SMInput) control. After washing, immunoprecipitated protein-RNA complexes were dephosphorylated and 3'-linker ligated on-bead to a custom oligonucleotide adapter. All samples (IPs and SMInputs) were heated in LDS Sample Buffer (LifeTech) containing 0.1 M DTT for 10 min at 70° and run on 4-12% NuPAGE Bis-Tris polyacrylamide gels in MOPS running buffer (LifeTech). Complexes were wet-transferred to iBLOT nitrocellulose membranes in NuPAGE transfer buffer (all LifeTech) containing 10% methanol (overnight, 4 °C, 30V). Successful immunoprecipitation was confirmed by performing standard western blotting on fractions (10%) of each sample. RNA-protein complexes in the range from expected molecular mass to 75 kDa above expected molecular mass (corresponding to the RBP crosslinked to RNAs of ~220 nucleotides in length) were excised from the membrane. RNAs were released by proteinase K treatment in the presence of urea and recovered by phenol/chloroform extraction and column purification (RNA Clean-Up kit; Zymo Research). Input samples were dephosphorylated and 3'-linker ligated to a custom oligonucleotide primer. All samples (IPs and SMInputs) were then reverse transcribed using AffinityScript reverse transcriptase (Agilent). After treatment with ExoSAP-IT (Affymetrix) and alkali, cDNAs were recovered by purification on Dynabeads MyONE Silane beads (LifeTech), 5'-linker ligated on-bead to a custom oligonucleotide primer, and again purified. cDNAs were quantified by qPCR analysis of a fraction of each sample using custom oligonucleotide primers specific to the 5' and 3' adapters. Half of the recovered cDNA was PCR-amplified (Q5 polymerase, NEB) using custom sequence-indexed oligonucleotide primers. PCR products were purified (Agencourt AMPure XP beads; Beckman Coulter), size-selected to 175-350 bp on 2% agarose gels, extracted (MinElute Gel Extraction kit; Qiagen), and quantified on a TapeStation using D1000 ScreenTape (Agilent). Libraries were sequenced on a HiSeq 4000 instrument (Illumina) in single-end 75 bp mode.
GEO
GSM4512279
GEO Accession
GSM4512279