GSM2945913
GSM2945913: HD109d100; Homo sapiens; RNA-Seq
Cells were manually scraped off of coverslips at time points 0, 60, 80, 100 and 130 of differentiation in PBS, spun down at 1000 RPM for 5 min and pellets were snap-frozen in liquid nitrogen. Frozen pellets were resuspended in 1 ml Trizol. 200 ml of chloroform was added, then samples were shaken and incubated at room temperature for 3 min prior to spinning at 13,000 RPM for 15 min. The clear supernatant was moved to a new tube and 318 ml of 100% ethanol was added. The supernatant was then transferred to an RNeasy column (Qiagen) and spun 30 sec at 13,000 RPM. Column was washed once with 500 ml buffer RW1 and spun for 30 sec at 13,000 RPM. 70 ml of buffer RDD and 10ul of RNase-free DNase (Qiagen) were added to column and incubated for 30 min. 500 ml of buffer RW1 was then added and column was spun at 13,000 RPM for 30 sec. Column was then washed again with 500 ml of RPE and spun at 13,000 RPM for 30 sec. The collection tube was changed and spun at 13,000 RPM for 1 min to remove residual buffer. 50 ml of RNase-free water was added and incubated at room temperature for 3 minutes before being spun into a clean collection tube at 13,000 RPM for 5 min. RNA was quantified using a nanodrop, then 500 ng was run on a 1% gel to ensure quality. At least 1,200 ng of RNA was submitted for sequencing. Library preparation and sequencing library construction were performed using the Illumina TruSeq Stranded mRNA library preparation kit. Briefly, total RNA samples were assessed for concentration using the Nanodrop 8000 (Thermo Scientific) and quality using the 2100 Bioanalyzer (Agilent). One microgram of total RNA per sample was used for poly-A mRNA selection using streptavidin-coated magnetic beads. cDNA was synthesized from enriched and fragmented RNA using reverse transcriptase (Super-Script II, Invitrogen) and random primers. The cDNA was further converted into double-stranded DNA, and the resulting dsDNA was enriched with PCR for library preparation. The PCR-amplified library was purified using Agencourt AMPure XP beads (Beckman Coulter). The concentration of the amplified library was measured with a NanoDrop spectrophotometer and an aliquot of the library is resolved on an Agilent 2100 Bioanalyzer. Sample libraries are multiplexed and sequenced on a NextSeq 500 platform (Illumina) using 75bp single-end sequencing. On average, about 20 million reads were generated from each sample.
GEO
GSM2945913
GEO Accession
GSM2945913