GSM8224369: MUT_D7_pM_NFIAn_rep1; Mus musculus; ATAC-seq
CaTS-ATAC was performed as previously described (Delás et al., 2023) with in-house produced Tn5. A dead cell removal step was introduced to enrich for live cells prior to fixation and transposition. The details are as follows: at the differentiation timepoint of collection, cells were washed once with PBS and dissociated with Accutase (Gibco, A1110501) for 5 min at 37°C. Cells were collected in 1.5 ml LoBind Eppendorf tubes (Eppendorf cat. #Z666548) and spun down at 400xg for 4 min. Cell suspensions were supplemented with LIVE/DEADTM Fixable Dead Cell Stain Near-IR fluorescent reactive dye (Thermo Fisher, L34976), according to the manufacturer's instructions (1 µl dye/1 ml PBS for 1x106 cells) and incubated for 30 min on ice protected from light. During the incubation, the autoMACS® Pro Separator (Miltenyi Biotec) was set-up according to the manufacturer's instructions. After the 30 min cell suspensions were divided into 15 ml falcon tubes and spun down for 10 min at 400xg at 4°C. Cells were resuspended, pooled into one tube, and counted. Cells were magnetically labelled with Dead Cell Removal Microbeads from the Dead Cell Removal Kit (Miltenyi Biotec, 130-090-101). For 1.0x107 cells, 100 µl beads were used. Cells and microbeads were mixed and incubated for 15 min at room temperature. If necessary, 1x Binding Buffer provided by the kit was added to the cell suspension to reach a minimum volume of 500 μl for separation. The samples were run through the autoMACS® Pro Separator (Miltenyi Biotec) using the 'Depl05' programme. The microbeads label dead cells, which are retained in the separation columns. Unlabelled live cells run through the column and were collected in a 15 ml falcon tube in PBS. Live cells were count and spun down for 4 min at 400xg at 4°C. Cell pellet was resuspended in 300 µl of PBS in 1.5 ml LoBind Eppendorf tubes. Cells were then fixed, transposed, stained for viability and intracellular markers (anti-SOX2-V450, anti-Nkx6.1-PE, goat anti-Olig2, rabbit anti-Nfia) and sorted. DNA of samples was isolated using the DNA Clean & Concentrator-5 kit (Zymo; D4013) as per manufacturer's instructions. Transposed DNA was first amplified with 2X PCR Master Mix NEB (Cat. No. M0541S), and 1 µl of constant forward primer and indexed reversed primer (Table S1) (both at 25 µM) in a total reaction volume of 50 ul. The program was as follows: 5 min at 72ºC, 30 s at 98ºC, followed by 7 cycles of 30 s at 98ºC, 30 s at 63 ºC and 60 s at 72 ºC, with a final extension of 5 min at 72 ºC. DNA was cleaned up with 1.8x volumes of AMPureXP beads (Beckman Coulter, Cat. No. A63882) and eluted in 18 µl of EB buffer. qPCR was carried out to determine the number of extra cycles using 2 µl of the amplified DNA in technical duplicates. Each 20 µl reaction contained 20 µl of 2X SYBR Green PCR assay, using PowerUP SYBR Green Master Mix (Thermo Fisher, Cat. No. A25742) and 2.5 µl of each primer (at 25 µM each). The qPCR program was as follows: initial activation for 2 min at 50ºC, 30 s at 98ºC, followed by 40 cycles of 10 s at 98ºC, 30 s at 63ºC and 60 s at 72ºC. The number of additional cycles was calculated as ¼ of the maximum amplification 86. The second amplification was done using 12.5 µl of amplified DNA, 2X PCR Master Mix NEB and 2.5 µl of each primer (at 25 uM) in a total volume of 50 ul. The program was the same as the first amplification without the initial extension and for the calculated number of cycles. Finally, re-amplified DNA was cleaned up using 1.8x volumes AMPureXP beads and eluted in 30 ul. Samples were quantified in the QuBit, size profiles examined in the Bioanalyzer using the DNA High Sensitivity DNA Kit (Agilent, Part No. 5067-4626).