GSM4980021: miCLIP HEK293T cells rep2; Homo sapiens; OTHER
For RNA extraction from HEK293T cells and mESCs, cells were washed in ice-cold PBS and collected on ice for the isolation of total RNA using the RNeasy Plus Mini Kit (Qiagen) following the manufacturer's recommended protocol. Poly(A)+ RNA was extracted using Oligo d(T)25 Magnetic Beads using the manufacturer's recommended protocol. Poly(A)+ concentration was measured using Qubit™ RNA HS Assay Kit (ThermoFisher Scientific). The quality of poly(A)+ RNA was ensured using High sensitivity RNA screen tapes for the 2200 Tape station system (Agilent). If a predominant peak for ribosomal RNA was still detectable an additional round of poly(A) selection was performed. Mouse heart RNA was purchased from Takara Clontech (order number 636202): RNA isolated by a modified guanidinium thiocyanate method followed by poly A+ RNA selection with two rounds of oligo(dT)-cellulose columns. RNA-seq: NGS library prep was performed with Illumina's TruSeq stranded mRNA LT Sample Prep Kit following Illumina's standard protocol beginning with fragmentation step (Part # 15031047 Rev. E). Libraries were prepared with a starting amount of 73ng for imb_koenig_2019_21 and amplified in 10 PCR cycles.Libraries were profiled in a DNA 1000 on a 2100 Bioanalyzer (Agilent technologies) and quantified using the Qubit dsDNA HS Assay Kit, in a Qubit 2.0 Fluorometer (Life technologies).  All 6 samples were pooled in equimolar ratio and sequenced on 1 NextSeq 500 Highoutput Flowcell, SR for 1x 84 cycles plus 1x 7 cycles for the index read. miCLIP2: Briefly, 1 µg of polyA selected and fragmented RNA was incubated with an m6A antibody [Synaptic Systems, 202 003] and crosslinked twice at 150 mJ/cm² at 254 nm followed by immunoprecipitation with protein A Dynabeads. After stringent washing, the 3' end of the RNA was dephosphorylated using T4 PNK and subsequently pre-adenylated L3-App linker was ligated. The 5' end was radioactively labelled and the samples were purified by SDS PAGE and transferred to a nitrocellulose membrane. After exposure to a Fuji film, the protein-RNA complexes were isolated and elution of the RNA was accomplished by proteinase K treatment, phenol-chloroform extraction and ethanol precipitation. Reverse transcription was perform for cDNA synthesis using RT primers against the L3-App linker. The cDNA was purified using MyONE Silane bead clean up system and the 5' linker was ligated. A pre-amplification PCR with 6 cycles was performed and subsequently the samples were size-selected using ProNex beads. To prevent overamplification of the library, the PCR cycles were optimised to a minimum. For the preparative PCR, the minimum amount of PCR cycles was used followed by a second size selection step using ProNex beads. Libraries were profiled in a High Sensitivity D1000 ScreenTape on a Tape station 2200 (Agilent) and quantified using the Qubit dsDNA HS Assay Kit, in a Qubit 2.0 Fluorometer (Life technologies). All samples in an experiment were pooled in equimolar ratio and sequenced on one NextSeq 500 High Output Flowcell, 75 cycle kit.