title: GSM4876528: Mouse_E6.5_ATAC-seq_gDNA; Mus musculus; ATAC-seq
library_construction_protocol: Fertilized chicken eggs (white leghorn; Gallus gallus domesticus) were obtained from a local breeder (LSL Rhein-Main) and incubated at 37°C and 80% humidity in a normal poultry egg incubator (Typenreihe Thermo-de-Lux). Following microsurgical procedures, the eggs were re-incubated until the embryos reached the desired developmental stages. The developmental progress was determined according to the staging system of HH (Hamburger and Hamilton, 1992). Mice were housed and bred under standard conditions in the CECAD ivRF. The breedings described were approved by the Landesamt für Natur, Umwelt, und Verbraucherschutz Nordrhein-Westfalen (LANUV), Germany (animal application 84-02.04.2015.A405). Tissue were extracted and resuspended to single cell level. ATAC-seq was essentially conducted following Buenrostro et al. Nat Methods 2013. In short, single cells were centrifugated at 5000 g for 5 min and supernatant was removed. Cells were then lysed with 100 µl cold lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0,1% IGEPAL CA-630) substituted with 4 µl of protease inhibitor (1 tablet/2 mL conc.) for at least 15 minutes on ice. Immediately after lysis, nuclei were centrifugated at 6000 g for 10 minutes at 4°C. Pellet was resuspended in transposase reaction mix (25µl 2x TD buffer, 10 µl transposase (Illumina), 15 µl nuclease-free H2O) and incubated for 30 min at 37°C. Finally sample was purified using Qiagen MinElute PCR purification kit according to the manufacturer's protocol.