GSM8536510: RNA-seq of RNA IP with an antibody specific for TMG; Homo sapiens; RIP-Seq
Total RNA was extracted with Trizol (Invitrogen) according to manufactor's recommendations. For RNA fragmentation, 20 ug of total RNA was incubated in 20 ul fragmentation buffer (10 mM Tris-HCl pH 7.4, 10 mM ZnCl2) at 94°C for 5 min. The fragmented RNA was then purified using the Monarch RNA cleanup kit (50 ug) (NEB) and 10 ug of RNA was immunoprecipitated with 3 ug IgG against TMG (MBL, RN019M) or non-specific IgG bound to protein A/G Dynabeads. Beads were washed three times with wash buffer (10 mM Tris-HCl, 150 mM NaCl, and 1% Empigen BB). RNA on magnetic beads was treated with FastAP (Thermo Fisher Scientific) at 37°C for 20 min and ligated to 20 pmole pre-activated 3′ adapter ( AppNNNNNCGTCGTGTAGGGAAAGAGTGTAGATddC) overnight at 37°C in RNA ligase buffer containing 15% PEG8000). RNA bound to beads was recovered by phenol/chloroform extraction and ethanol precipitation. The RNA-adapter hybrid was converted into complementary DNAs with an RT primer (5' phosphate-CAGCAGGAATGCCGAGACCGATCTACACTCTTTCCCTACACGA ), size-fractionated by electrophoresis on a 12% polyacrylamide gel containing 8 M urea and circularized by circLigase ssDNA ligase (Biosearch technologies). Circularized cDNAs were PCR-amplified with PCR primers containing demultiplexing codes (AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACG, and CAAGCAGAAGACGGCATACGAGAT[5nt code)TAGCTGATGCTACGACCACAGG)  to be converted into linear DNA fragments adequate for Illumina high-throughput DNA sequencers. The samples were sequenced on Illumina NextSeq 500 Sequencer with a primer (AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACG) to obtain 76-nt reads and with the second primer (CCTGTGGTCGTAGCATCAGCTA) to obtrain demultiplixing codes. The raw image data was transformed by base-calling into sequence data and stored in FASTQ format.