GSM8198004: ATAC_Candida_2h_R2_1; Mus musculus; ATAC-seq
At the end of the treatment period, cells were washed with PBS and then scraped in 0.5 ml PBS per million cells. 50,000 cells were directly used for ATAC-seq. Cells were centrifuged at 4 °C and 500 rcf for 5 minutes. The supernatant was removed, and cells were lysed and tagmented at 37 °C for 30 minutes in the following mixture (per sample): 9.75 µl water, 2x transposase buffer (Illumina cat. No. 20034197), 0.5 µl PIC (Roche cat. No. 11873580001), 2 µl TDE1 DNA Enzyme (Illumina cat. No. 20034197), and 0.25 µl 1% Digitonin (Promega cat. No. G9441). The DNA was cleaned using the Qiagen MinElute kit (Qiagen cat. No. 28006) according to the manufacturer's recommendations and eluted in 12 µl EB buffer. To estimate the enrichment cycle number, a qPCR was set up using 2.9 µl water, 0.5 µl index primer 1 noMX and Index primer 2.1 barcode, respectively, 0.1 µl 100x SYBR green (Invitrogen cat. No. S7563), 5 µl NEBnext HF 2x MM (NEB cat. No. M0541L), and 1 µl eluted sample, and run as follows: 5 min at 72 °C, 30 seconds at 98 °C, 25x cycles of (10 sec at 98 °C, 30 sec at 63 °C and 1 min at 72 °C). The cycle number was determined based on the amplification entering the early exponential phase. Subsequently, an enrichment PCR was set up with 10 µl water, 2.5 µl Index primer 1 noMX and Index primer 2 barcode, respectively, 25 µl NEBnext HF 2x MM (NEB cat. No. M0541L), and 10 µl eluted sample (see Buenrostro et al. 2013 for primer sequences https://doi.org/10.1038/nmeth.2688). A left-sided 1.6x clean-up was performed using AMPure XP beads (Beckman Coulter cat. No. A63881). To that end, 80 µl beads were added and the sample incubated for 10 minutes at room temperature, followed by 10 minutes on a magnetic rack at room temperature. The beads were washed twice with 80% EtOH, air-dried for 2 minutes and the DNA eluted in 50 µl EB buffer. A second 0.5/1.4 double-sided clean-up was performed. 25 µl beads were added and the sample incubated 10 minutes at room temperature followed by 10 minutes on a magnetic rack as before. The supernatant was transferred to a new well and mixed with additional 45 µl beads, and the left-sided clean-up continued as described above. The DNA was eluted in 15 µl EB buffer