shMETTL3_GSC_RIP_replicate2
A library was prepared from each RNA sample. Five nanogram of each m6ARIP sample were used. As a control, a library was prepared also from 10ng of the respective polyA-RNA sample used for m6A RIP enrichment. The NEBNext RNA Ultra II library prep kit (#E7770S, NEB) was applied and samples were prepared according to the manufacturers instruction with slight modifications. The fragmentation step was omitted and the fragments were purified using QIAQuick column (#28104, Qiagen, Germany) after second-strand synthesis. Index primers of NEBNext Multiplex oligonucleotides for Illumina (Index Primers Set 1) were used (E7335S, NEB). The final PCR enrichment of adaptor-ligated libraries was performed using 12 cycles. Library quality was confirmed on a Tapestation2200 (Agilent Tech) and the DNA concentration was quantified with the Qubit dsDNA HS kit (Q32851). Volumes corresponding to 2 nM DNA were pooled and sequenced on a HiSeq2000 (Illumina) with 50bases single-end read.