title: GSM4123923: CHIR rep2; Homo sapiens; RNA-Seq
library_construction_protocol: QIAshredder and RNeasy Mini Kit (Qiagen, UK) was used to isolate RNA from transfected U2OS cells according to the manufacturer's protocol. Integrity of RNA was determined using a bioanalyzer instrument (Agilent Technologies, Santa Clara, CA, USA). A cDNA library including 5 replicates per condition was prepared using the TruSeq Stranded mRNA Sample Prep Kit (Illumina Inc., San Diego, CA) according to the manufacturer´s recommendation. Briefly, 1µg of total RNA was used for first-strand synthesis performed with a random hexamer and SuperScript II (Life Technologies, Carlsbad, CA, USA). Second-strand synthesis was performed using dUTP and the Illumina-specific Second Strand Marking Master Mix. After end repair and A-tailing indexed adaptors were ligated to the cDNA fragments. Fragments successfully ligated with adaptor molecules on both ends were enriched by PCR for 15 cycles and purified with AMPure XP Beads (Beckman Coulter Inc., Brea, CA). The final libraries were quality checked on an Agilent Bioanalyzer and quantified with qPCR using a commercially available PhiX-library (Illumina Inc., San Diego, CA) as a reference. All samples were run in five biological replicates and a total of 30 equimolarily pooled samples were sequenced two High Output flow cells on an Illumina NextSeq in a paired end run with 2x 75 cycles.
identifiers.namespace: GEO
identifiers.id: GSM4123923