title: GSM4683236: C6_calb_live_24hrs_250ng; Homo sapiens; RNA-Seq
library_construction_protocol: At 4 and 24 hours cells were lysed with RLT buffer. Prior to subjection to the RNeasy Mini Kit (Qiagen), lysates were homogenized using a 1 mL syringe with a 0.8 x 15 mm needle. RNA was subsequently extracted following manufacturers' protocol, including an on-column DNAse digestion using the RNAse-Free DNAse set (Qiagen). Quantification and quality assessment of extracted RNA was performed using the Qubit RNA HS assay (Thermo Fisher Scientific) and Agilent 2200 TapeStation (RNA HS Screentape, Agilent), respectively. The majority of samples subjected to quality assessment revealed an RNA integrity number (RINe) of  8. Libraries were generated from the extracted RNA using the QuantSeq 3' mRNA-Seq Library Prep Kit-FWD from Lexogen (Lexogen, Vienna, Austria) in accordance to the manufacturers' protocol. Three separate preparations were performed, split by PBMC donor, in turn limiting the number of samples to 14 to 18 samples per prep. RNA input was normalized to 100 ng for donor A, and to 250 ng for donors B and C. An aliquot (1:10) of double stranded cDNA libraries was used for quantitative PCR, in turn indicating 17 – 18 cycles as optimal for endpoint PCR (17- donor B; 18 - donors A, C). Accurate quantification and assessment of quality of the generated libraries was performed using Qubit dsDNA HS assay (Thermo Fisher Scientific, Waltham, USA) and Agilent 2200 TapeStation (HS-D1000 ScreenTape, Agilent, Santa Clara, USA). The cDNA concentration and average fragment size were used to determine the molar concentration of the individual libraries. Consequently, libraries were pooled equimolar to 100 fmol. After a final dilution of the pool to a concentration of 4 nM, the libraries were sequenced on a NextSeq 500 instrument (Illumina, San Diego, USA), with 75 cycle (i.e. 75bp single-end sequence reads), high output kit with a 1.1 pM final loading concentration.
attributes.tag: GEO Accession
attributes.value: GSM4683236