title: GSM1143925: KYSE70 shSOX2(1) Day 4; Homo sapiens; RNA-Seq
library_construction_protocol: For each sample, polyA+ RNA was isolated from 5 ug of total RNA using Dynabeads mRNA DIRECT Kit (Ambion). RNA was fragmented by incubation at 98 °C for 41 min in 1× RNA restoration buffer (Ambion). cDNA was synthesized with 20-40 ng of polyA+ RNA with a peak length around 600-700 bases with the use of SuperScript III Reverse Transcriptase, SuperScript Double-Stranded cDNA Synthesis kit, and random primers (Invitrogen). Primers were annealed at room temperature for 10 min followed by first strand synthesis for 1 h at 50°C and second strand synthesis for 2.5 h at 16°C. Following cDNA size selection with 1.8× AMPure XP beads (Beckman Coulter), end repair, and A-tailing, adapter ligation was performed using the TruSeq DNA Sample Preparation kit (Illumina) according to manufacturer’s instruction. Adaptor-ligated cDNAs were cleaned up with 0.8× AMPure XP beads followed by 8 cycles of PCR with primers complementary to the adaptor sequences with indexes. The PCR products were purified with 0.8× AMPure XP beads and then subject to HiSeq 2000 sequencing.
attributes.tag: GEO Accession
attributes.value: GSM1143925