title: GSM3474011: 3A_DS_ICD: DSCAM-ICD-YFP-HA, replicate A; Homo sapiens; RNA-Seq
library_construction_protocol: 5 million cells per condition were lysed and homogenized using Trizol reagent (Ambion, Life Technologies) and subsequent phase separation was performed using Chloroform (Sigma) following centrifugation according to the Trizol reagent protocol. Total RNA was isolated using the PureLink RNA Mini Kit (Ambion, Life Technologies) according to the manufacturers' user manual. In brief, the aqueous layer containing the RNA was mixed with 70% (v/v) ethanol and bound to an RNaesy column (Ambion, Life Technologies). Genomic DNA was removed by on-column DNAse treatment using PureLink DNAse (Life Technologies). Subsequently, the RNA was washed and then eluted in non-DEPC treated nuclease free water and stored in aliquots at –80 °C until further processing. RNA concentration and purity were determined spectrophotometrically using the Nanodrop ND-1000 (Nanodrop Technologies) and RNA integrity was assessed using a Bioanalyser 2100 (Agilent). Per sample, an amount of 2ug of total RNA was used as input. Using the Illumina TruSeq® Stranded mRNA Sample Prep Kit (protocol version Rev E, October 2013) poly-A containing mRNA molecules were purified from the total RNA input using poly-T oligo-attached magnetic beads. In a reverse transcription reaction using random primers, RNA was converted into first strand cDNA and subsequently converted into double-stranded cDNA in a second strand cDNA synthesis reaction using DNA PolymeraseI and RNAse H. The cDNA fragments were extended with a single 'A' base to the 3' ends of the blunt-ended cDNA fragments after which multiple indexing adapters were ligated introducing different barcodes for each sample. Finally, enrichment PCR was carried out to enrich those DNA fragments that have adapter molecules on both ends and to amplify the amount of DNA in the library. Sequence-libraries of each sample were equimolarly pooled and sequenced on an Illumina NextSeq 500 instrument (High Output, 75 bp, Single Reads, v2).
attributes.tag: GEO Accession
attributes.value: GSM3474011