GSM3576228
GSM3576228: 16B_MEFs; Homo sapiens; RNA-Seq
Transduced and untransduced H1-iCas9 cells were lysed on ice using RLT Plus buffer, and total RNA was extracted with the RNeasy Plus Mini Kit (Qiagen). Following lysis, supernatant was transferred to a QIAShredder spin column (Qiagen) and centrifuged at maximum speed for 2 minutes. The supernatant was transferred to RNeasy spin columns and on-column DNase digestion was performed using the RNase-Free DNase set (Qiagen). RNA extraction procedure was continued according to manufacturer's instructions. Samples were stored at -80ºC prior to sequencing. Total RNA was quantified using Qubit RNA BR Assay fluorescent chemistry (ThermoFisher Scientific) and diluted to 1ng/µl. 1µL of the dilution stock was used to obtain RNA Integrity Number (RIN) using the Agilent Bioanalyzer RNA 6000 Pico kit (Agilent). Lowest RIN was 8.4, median RIN score was 9.7. 750ng per sample was processed according to Illumina TruSeq Stranded mRNA sample preparation protocol (Part # 15031047 Rev. E). 1µL top stock of each purified final library was run on an Agilent Bioanalyzer dsDNA High Sensitivity chip (Agilent). The libraries were quantified using Qubit dsDNA HS Assay Kit (ThermoFisher Scientific) and were pooled at equimolar ratios after size adjustment. The final pool was run on an Agilent Bioanalyzer dsDNA High Sensitivity chip and quantified using NEBNext Library Quant Kit for Illumina (NEB). The quantified pool was hybridized at a final concentration of 2.2pM and sequenced single-end on the Illumina NextSeq500 platform using high-output v2.5 75c chemistry.
GEO
GSM3576228
GEO Accession
GSM3576228