GSM8403035: D2_CC_1; Mus musculus; ATAC-seq
Nuclei pellets were resuspended gently in transposition reaction mix (25 µL 2X TD Buffer [Illumina Cat #FC-121-1030], 2.5 µL transposase [Illumina Cat #FC-121-1030] and 22.5 µL nuclease-free water) and incubated at 37℃ for 30 min followed immediately with purification using Qiagen MinElute kit whereby transposed DNA were eluted in 10 µl Elution Buffer. Transposed DNA fragments were amplified initially using 1X NEBnext PCR master mix (New England Labs Cat #M0541), 25 µM customized Nextera PCR primers 1 and 2 and 0.6% final concentration of 100X SYBR Green I (Invitrogen Cat #S-7563) in a thermocycler with the following parameters: 72℃ for 5 min; 98℃ for 30 sec; 5 cycles of 98℃ for 10 sec, 63℃ for 30 sec; and 72℃ for 1 min. A side qPCR amplification using a small aliquot of the amplified DNA was used to determine the necessary additional number of cycles to stop amplification prior to saturation (1/4 of max fluorescent intensity) to reduce GC and size bias. The remaining DNA samples were amplified to the correct number of cycles and then purified and eluted using Qiagen PCR Cleanup kit in 20 µl of Elution buffer. Library quality control (QC) was performed using Agilent Bioanalyzer High Sensitivity DNA chip with the samples diluted 1:5.