title: Illumina NovaSeq 6000 paired end sequencing; Whole Genome Sequencing of MCF10A cells
design: Whole Genome Sequencing of MCF10A cells
library_construction_protocol: After 7 day in culture, MCF10A cells were harvested from T75 flask. Media was removed and cells were washed with 5ml sterile PBS. 3ml of trypsin/EDTA was added and cells were incubated for 10 min at 37oC. Once cells were detached, 5ml of media was added and cells were resuspended to achieve single cells solution. 7ml of cell suspension was transferred to a 15ml Falcon tube and remove a 10ul aliquot was removed for counting.14x10^6 cells were centrifuged at 450g for 5min, washed with 5ml of PBS and centrifuged a second time to pellet cells prior to removal of PBS. The cell pellet was then stored on ice before immediate processing for extraction of genomic DNA. Purification of genomic DNA was performed using the QIAamp Blood Maxi Kit (Qiagen) according to manufacturer's instructions and DNA concentration was determined using the Qubit DNA Assay Kit and a Quibit2.0 Fluorometer (ThermoFisher) Library preparation was performed by Novogene. The genomic DNA was randomly fragmented by sonication to the size of 350 bp, then DNA fragments were end polished, A-tailed, and ligated with the full-length adapters of Illumina sequencing, and followed by further PCR amplification. The PCR products as the final construction of the libraries were purified with AMPure XP system. Then libraries were checked for size distribution by Agilent 2100 Bioanalyzer (Agilent Technologies, CA, USA), and quantified by real-time PCR (to meet the criteria of 3 nM).