title: GSM8241809: E10.5_PGC_Male_TRIM28KO_ATAC_2; Mus musculus; ATAC-seq
library_construction_protocol: PGCs from dissected embryonic gonads (E12.5, E13.5, E14.5) and dissected embryo trunks (E10.5, E11.5) were dissociated in 0.05% Trypsin in 5% CO2/37C for 5 minutes. Digestions was quenched using 10% FBS in DMEM High Glucose media and cells were pelleted at 1200 rpm x 5 minutes. Resulting pellet was resuspended in 10% FBS, High Glucose DMEM + DNAse and treated for 5 minutes at 5%CO2/37C and pelleted by centrifugation at 1,200 RPM for 5 minutes. PGCs were then suspended in DPBS + 1% BSA and GFP+, 7AAD- cells were collected in DPBS + 1% BSA. . Libraries were prepared according to the protocols published in Corces et al., Nature Methods 2017 (DOI: https://doi.org/10.1038/nmeth.4396) Briefly: Samples were spun down at 750 r.c.f at 4C for 10 minutes, resuspended in buffer RSB and spun again at 750 r.c.f at 4C for 10 minutes. After last centrifugation, buffer RSB was removed and Tn5 Enzyme mix added. Samples were treated at 37C for 30 minutes, shaking at 1000 rpm. Reaction was immediately quenched on ice with ice-cold PBS. DNA was purified using NEB Monarch DNA Purification kit according to the manufacturer's instructions. Purified DNA was used as a template and Nexteera indexing adapters added to the reaction with NEB Next High fidelity PCR mix. Samples were run for 5 cycles, split and run for an additional 4 and 8 cycles. Resulting libraries were purified using Agencourt XP beads and quantified using the Quibit HS DNA quantification kit according to manufacturer's instructions. Libraries were sequenced on an Illumina NovaSeq 6000 (E14.5) or Illumina NovaSeq X Plus (E10.5).