GSM4007036: BL3_rep1; Bos taurus; RNA-Seq
Cells were seeded in 3 biological replicates at a density of 2.5x105 cell/ml. RNA extraction was performed using the PureLink RNA Mini Kit (Life technologies, catalogue number 12183018A) following the manufacturer's instructions. Briefly, cells were pelleted, lysed and homogenized using a 21-gauge needle, then 70% ethanol was added to cell lysates and samples loaded onto spin cartridges to bind RNA. After 3 washes RNA was eluted in RNase-free water. The quality of extracted RNA was verified using a Bioanalyzer 2100 and quantification carried using Qubit (Invitrogen, catalogue number Q10210). Strand-specific RNA-sequencing (ssRNA-seq) libraries were prepared using the Illumina Truseq Stranded mRNA Sample Preparation Kit (Illumina, catalogue number RS-122-2101) following the manufacturer's instructions. Briefly, 1ug of total RNA was used to purify mRNA using poly-T oligo-attached magnetic beads. mRNA was then fragmented and cDNA was synthesized using SuperScript III reverse transcriptase (Thermofisher, catalogue number 18080044), followed by adenylation of the 3'-end, barcoding and adapter ligation. The adapter ligated cDNA fragments were then enriched and cleaned with Agencourt Ampure XP beads (Agencourt, catalogue number A63880). Libraries validation was conducted using the 1000 DNA kit on 2100 Bioanalyzer (Agilent Technologies, catalogue number 5067-1504) and quantified using qubit (Thermofisher, catalogue number Q32850). ssRNA libraries were sequenced on Illumina Hiseq2000 and Hiseq4000.
GEO Accession
GSM4007036