title: GSM7245004: iCLIP-Seq of MYNN, replicate 1; Homo sapiens; RIP-Seq
library_construction_protocol: 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 10ug 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. miCLIP procedure was performed as decribed. Total RNA was extracted from HEK293 cells using TRIzol as per manufacturer's protocol. RNA was treated with DNase I to eliminate any contaminating genomic DNA. We used 12 μg of total RNA for miCLIP-seq. 2 μL of RNA Fragmentation Reagents was added to the RNA suspended in 20 μL of sterile water. The mixture was incubated in a heat block at 75°C for exactly 12 minutes. 2.2 μL of stop solution was added to stop the reaction. The fragmented RNA was incubated with 10 μg of m6A antibody for 2 hours at 4°C. RNA-antibody complexes were UV crosslinked twice with 0.15 J/cm2 at 254 nm in a Stratalinker 1800.  Protein G Dynabeads (100 μL) were added, and the samples were rotated for 2 hours at 4°C. Remaining steps, including dephosphorylation, adapter ligation, SDS-PAGE, and RNA-extraction are similar to the iCLIP procecure. 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.