title: GSM2416960: MC903 Rep4; Mus musculus; RNA-Seq
library_construction_protocol: Samples of murine skin were obtained and stored in RNAlater (Sigma) before processing following the manufacturer’s instructions. To extract whole tissue RNA, samples were homogenized using a bead homogenizer and processed using the Qiagen RNeasy kit following the manufacturer’s instructions. Following total RNA extraction, samples were treated with DNase (Turbo DNA-Free Kit, Thermo Scientific) following the manufacturer’s instructions before sequencing library preparation. Library preparation was performed with 1 ug of total RNA, integrity was determined using an Agilent Bioanalyzer. Ribosomal RNA was removed by a hybridization method using Ribo-ZERO kits (Illumina-EpiCentre). mRNA was then fragmented in buffer containing 40 mM Tris Acetate (pH 8.2), 100 mM Potassium Acetate, and 30mM Magnesium Acetate and heated to 94°C for 150 seconds. mRNA was reverse transcribed to yield cDNA using SuperScript III RT enzyme (Life Technologies, per manufacturer’s instructions) and random hexamers. A second strand reaction was performed to yield ds-cDNA. cDNA was blunt ended, had an A base added to the 3’ ends, and then had Illumina sequencing adapters ligated to the ends. Ligated fragments were then amplified for 12 cycles using primers incorporating unique index tags. Fragments were sequenced on an Illumina HiSeq-3000 using single reads extending 50 bases.
attributes.tag: GEO Accession
attributes.value: GSM2416960