GSM3070505: T30min; Homo sapiens; RNA-Seq
Genomic DNA was isolated from samples of a minimum of 2E7 cells using the Blood and Cell Culture Midi Kit (Qiagen cat. 13343, Valencia, CA) 5µl amplicon or 1µl diluted plasmid library was used as template in 13 50µl Herculase II DNA polymerase reactions per sample to attach pooled variable-length spacers and Illumina indexes (custom primers, see manuscript). 24 cycles were used to amplify DNA in the first and second PCR, respectively. The amplicon fragments after PCR 2 comprise a 354-362bp library with variable 20bp sgRNA sequence in the middle. DNA was pooled by sample and purified using the Nucleospin Gel and PCR Clean-up kit (Clontech cat. 740609.250, Mountain View, CA). DNA was quantified using a Qubit high-sensitivity DNA quantification assay (Thermo Fisher cat. Q32851) and Take3 microspot plate reader (BioTek). DNA quality was analyzed by Experion CHIP assay (BioRad cat. 7007-163, Hercules, CA). Clusters were generated on the flow cell using the HiSeq Rapid Duo CBot Sample Loading Kit (Illumina CT- cat. 403-2001, San Diego, CA). A single-read rapid run of 75 cycles was performed on a HiSeq 1500 (Illumina cat. GD-402-4002) using the HiSeq Rapid SBS kit (Illumina cat. FC-402-4022) with 10% PhiX.
GEO Accession
GSM3070505