GSM5139882: MuFL 6/3 MA4 LMPP; Mus musculus; ATAC-seq
cells were sorted from freshly isolated mouse tissues or previously frozen human tissues; single cell preparations were stained with cell surface markers and directly sorted into transposition buffer Following the discarding of supernatant the nuclei pellet was resuspended in 50 μl transposition mix with the following Tn5 volumes according to cellular input (1000-5000 (0.5 μl Tn5), 5000-10,000 (1.0 μl Tn5), 10,000-15,000 (1.5 μl Tn5) with the remainder of the total reaction adjusted with water (25 μl 2× TD buffer, x μl transposase, 16.5 μl PBS, 0.5 μl 1% digitonin, 0.5 μl 10% Tween-20, x μl H2O) and incubated at 37 °C for 30 min in a thermomixer with 1,000 rpm shaking. All reactions were cleaned up using the Zymo DNA Clean and Concentrator-5 Kit (Cat# D4014) with DNA eluted in 11 μl of elution buffer and PCR amplified with the NEBNext Hi-Fidelity PCR Master Mix (Cat# M0541L) with a custom dual barcoding strategy as previously described (Mezger et al. PMID: 30194434). The amplification reaction was stopped at 5 cycles and 5 μl of the pre-amplified mixture was run in a 15 μl qPCR reaction to determine the optimal number of additional cycles that corresponded to 1/3 of the maximum fluorescent intensity up to a total of 13 cycles (Buenrostro et al. PMID: 25559105). A final clean up was performed, libraries were eluted in 20 μl and quantified using the NEBNext Library Kit for Illumina (Cat# E7645L), assessed using the Agilent Bioanalyzer High Sensitivity DNA Analysis kit (Cat# 5067-4627) and finally double size selected with the Zymo Select-a-Size DNA Clean & Concentrator kit (Cat# D4084).