GSM4386543: N1177; Mus musculus; RNA-Seq
RNA was isolated as described previously (Walker et al., 2018) using RNAeasy Mini Kit (Qiagen, Fredrick, MD) with a modified protocol from the manufacturer allowing for the separation and purification of small RNAs from total RNA. Briefly, after cell lysis and extraction with QIAzol (Qiagen, Fredrick, MD), small RNAs were collected in the flow-through and purified using the RNeasy MinElute spin columns and total RNA was purified using RNeasy Mini spin columns. Samples were treated with DNAse to rid them of genomic DNA and run on nanodrop and an Agilent Bioanalyzer 2100 to confirm RNA purity, integrity and concentration. Libraries were prepared using the TruSeq Stranded mRNA HT Sample Prep Kit protocol (Illumina, San Diego, CA). Briefly, poly A selection and fragmentation of 300 ng of RNA was performed, and the resulting RNA was converted to cDNA with random hexamers. Adapters were ligated and samples were size-selected with AMPur XP beads (Beckman Coulter, Brea, CA). Barcode bases (6 bp) were introduced at one end of the adaptors during PCR amplification steps. Library size and concentration were assessed using Tape Station (Life Technologies, Grand Island, NY) before sequencing. Libraries were pooled for multiplexing (pool of 21 samples) and sequenced on a HighSeq2500 System using V4 chemistry with 50 base pair single-end reads at UCLA Neuroscience Genomics core. Each pool was sequenced 3 times with the goal of obtaining ~30 million reads per sample.
GEO Accession
GSM4386543