GSM4876828: CLIP_MUT_MA_2; Mus musculus; RIP-Seq
Dissected whole striata were stored in RNAlater (ThermoFisher) following manufacturer's instructions to stabilize the RNA and protein followed by cross-linking immunoprecipitation library preparation. Prior to crosslinking, a portion of the pooled tissue from each replicate was removed and stored in -80C for later RNA extraction and bulk RNA-seq library preparation. Following the completion of membrane transfer, the membrane was cut with a clean razor to obtain a vertical membrane slice per lane from 50 kDa (molecular weight of hnRNP H) to 75 kDa, which translates to 30 to 70 nucleotide RNA fragments crosslinked to the protein. RNA was then extracted from the membrane slices following a previously described procedure (Rieger et al., 2018). The same extraction procedure (starting with the addition of 7M urea) was used to isolate RNA from samples previously stored for total RNA-seq. Following RNA extraction, sample concentration was quantified with Agilent Bioanalyzer and approximately 0.2 ng of RNA was used to prepare next-generation sequencing libraries following a protocol for library preparation (Rieger et al., 2018; BioRxiv). Because the RNA adapter on each sample contained a unique barcode, the cDNA libraries generated from the sample (12 CLIP-seq and 12 RNA-seq libraries) were multiplexed and pooled for increased throughput. A pooled library at a concentration of 10 nM were shipped to University of Chicago Sequencing core and subjected to 100 bp paired-end 2 x 100 sequencing in a single lane on Illumina HiSEQ4000. To increase read coverage for the RNA-seq samples, those 12 cDNA libraries were pooled (10 nM concentration) for sequencing for a second time in one lane using Illumina HiSEQ4000. CLIP-seq and RNA-seq