GSM4063664: Undiff MS2control miTRAP 3; Mus musculus; miRNA-Seq
miTRAP eluates were mixed with 80 µl of nuclease-free water, 80 µl ammonium acetate solution (5 M; Sigma-Aldrich) and 400 µl phenol/chloroform (5:1, pH 4.5; Sigma-Aldrich). On centrifugation at 15 000g for 10 min, 370 µl of upper phase was transferred to new tube and precipitated with 1 ml ethanol and 2 µg glycogen (Roche). RNA pellets were dissolved in 24 µl of nuclease-free water. Input RNA was phenol–chloroform extracted from 200 µl cell extract upon Proteinase K treatment (20 mg/ml; Life Technologies). Input RNA was dissolved in 30 µl of nuclease-free water. According to Braun et al, 2014 (PMID: 24510096). RNA prepared from total miTRAP eluates and 8% input RNA (500 ng) was used in the small RNA protocol with the TruSeq™ Small RNA Sample Prep Kit v2 (Illumina, San Diego, CA, USA) according to the instructions of the manufacturer. The barcoded libraries were size restricted between 140 and 165 bp, purified and quantified using the Library Quantification Kit-Illumina/Universal (KAPA Biosystems, Woburn, MA, USA) according to the instructions of the manufacturer. A pool of up to 10 libraries was used for cluster generation per lane. Library DNA at a concentration of 10 pM was clustered using an Illumina cBot according to the SR_Amp_Lin_Block_Hybv8.0 protocol of the manufacturer. Sequencing of 50 bp was performed with an Illumina HighScan-SQ sequencer using version 3 chemistry and the version 3 flowcell according to the instructions of the manufacturer.
GEO
GSM4063664
GEO Accession
GSM4063664