GSM5649849: E12_Input_Rbm38; Mus musculus; OTHER
Lysates were treated with Turbo DNase (Thermo Fisher Scientiﬁc) and RNase I (Thermo Fisher Scientiﬁc) diluted 1/250 in PBS for 5 minutes at 37°C to digest genomic DNA and partially digest RNA to short fragments of an optimal size range. RNA-protein complexes were puriﬁed using 100 mL of protein G Dynabeads (Thermo Fisher Scientiﬁc) and 5μg of anti-Flag M2 (Sigma-Aldrich) antibody, with 2% (20ul) of the total lysate retained for use as a size-matched input (SMI) control. SMI samples were processed as previously described (Van Nostrand et al., 2016). Following stringent high salt washes, the immunoprecipitated RNA was 3′ end dephosphorylated and ligated to a pre-adenylated adaptor at the 3′ end, as described previously. After extensive additional washes and buffer exchange, the immunoprecipitated RNA was 5′ end-labeled using ATP [γ-32P]. The puriﬁed protein-RNA complexes and SMI control samples were separated with SDS-PAGE and transferred to nitrocellulose membrane. Protein was digested using proteinase K, the RNA was puriﬁed using phenol/chloroform. Purified RNA was reverse transcribed into cDNA. The cDNA was size selected by resolving samples on a 6% TBE-Urea gel, circularized to add the adaptor to the 5′ end, digested at the internal BamHI site, and then PCR ampliﬁed using AccuPrime SuperMix I (Thermo Fisher Scientiﬁc). The ﬁnal PCR libraries were puriﬁed by agarose gel electrophoresis using gel extraction columns (QIAGEN). The eluted DNA from the low, middle, and high fractions was pooled together and submitted for sequencing.