GSM2611673: MEF before NT; Mus musculus musculus x M. m. castaneus; ATAC-seq
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
ATAC-Seq
Antigen
ATAC-Seq
Cell type
Cell type Class
Embryonic fibroblast
Cell type
MEF
Tissue
Embryonic Fibroblast
Lineage
primaryCells
Description
Mouse Embryonic Fibroblast
Attributes by original data submitter
Sample
source_name
MEFs
cell type
Cultured mouse embryonic fibroblasts were permeabilized for nuclear transfer
library strategy
ATAC-seq
strain
MEFs were established using embryos derived from Musculus musculus crossed with Mus musculus castaneus
Sequenced DNA Library
library_strategy
ATAC-seq
library_source
GENOMIC
library_selection
other
library_construction_protocol
Cultured cells were harvested, washed with PBS, and then transferred to cold lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3mM MgCl2, 0.1% IGEPAL, 0.5% Triton X-100). Triton X-100 was needed especially for permeabilizing cells after nuclear transfer. Cells in lysis buffer were collected by centrifugation and the cell pellet was resuspended in the transposon reaction mix (25 μl 2x TD Buffer [Illumina, FC-121-1030], 2.5 μl Tn5 Transposes [Illumina, FC-121-1030], 22.5 μl Nuclease Free H2O). A total of 50 μl of the transposon reaction was used for 50,000 cells while 3,000 cells were incubated in 10 μl of the transposon reaction. Transposition reaction was performed at 37oC for 30 min. After the transposition reaction, transposed DNA was purified using a QIAGEN MinElute kit and used for the subsequent PCR amplification. Cultured cells with 1,000 were directly subjected to PCR amplification without using the purification kit. Transposed DNA was amplified by two rounds of PCR using NEB Next High Fidelity Master Mix (New England Labs, M0541) and the Customized Nextera PCR primers totaling 50 μl (Buenrostro et al., 2013). The first PCR cycles varied from 5 to 15 cycles depending on the starting cell numbers. The number of second PCR cycles was determined by qPCR using the 10% of the total PCR reaction in order to amplify the DNA in the exponential growth phase. The amplified DNA was purified using QIAGEN PCR Cleanup kit and finally resuspended in 20 μl of elution buffer. The quality of library was checked on polyacrylamide gels and quantified using the KAPA library quantification kit for Illumina sequencing platforms (KAPA Biosystems).