GSM6755004: MCF7.E.print.72hr; Homo sapiens; RNA-Seq
We added 1mL of 5mg/mL Dispase to each ring and incubated for 20 minutes at 37C. After the matrix was digested, cells were collected in media and spun at 1500xg for 5 minutes. The supernatant was aspirated and 45mL of cold PBS was added gently to the tube. We spun the tube at 2000xg for 5 minutes, and the supernatant was aspirated. The cell pellets were frozen quickly by placing tubes at -80C. Libraries for RNA-Seq were prepared with KAPA Stranded mRNA-Seq Kit (Cat.KK8420). The workflow consists of mRNA enrichment and fragmentation, first strand cDNA synthesis using random priming followed by second strand synthesis converting cDNA:RNA hybrid to double-stranded cDNA (dscDNA), and incorporates dUTP into the second cDNA strand.  cDNA generation is followed by end repair to generate blunt ends, A-tailing, adaptor ligation and PCR amplification. Different adaptors were used for multiplexing samples in one lane. Sequencing was performed on Illumina NovaSeq 6000 for PE 2x150 run. Data quality check was done on Illumina SAV. Demultiplexing was performed with Illumina Bcl2fastq v2.19.1.403 software.