title: GSM4197233: Germline_YA_ATAC_se_rep2; Caenorhabditis elegans; ATAC-seq
library_construction_protocol: Frozen worms were broken by smashing using a Biopulverizer then the frozen powder was thawed in 8 ml Egg buffer (25 mM HEPES pH 7.3, 118 mM NaCl, 48 mM KCl, 2 mM CaCl2, 2 mM MgCl2). Ground worms were pelleted by spinning at 800 g for 3 min, then resuspended in 8 ml of Buffer A (0.3 M sucrose, 10 mM Tris pH 7.5, 10 mM MgCl2, 1 mM DTT, 0.5 mM spermidine 0.15 mM spermine, protease inhibitors (Roche complete, EDTA free) and 0.025 % IGEPAL CA-630). The sample was dounced (two strokes) in a 14-ml stainless steel tissue grinder (VWR), then the sample spun 100 g for 6 min to pellet remaining worm fragments. The supernatant was kept (nuclei batch 1) and the pellet resuspended in a further 7 ml of Buffer A and dounced for 30 strokes. This was spun 100 g for 6 min to pellet debris and the supernatant was kept (nuclei batch 2). This way, we found that the first fraction was already enriched for germline nuclei while the second fraction was enriched for somatic nuclei. At this stage, nuclei quality was assessed by microscopy before proceeded to immuno-staining. Nuclei immuno-staining was performed on the appropriate fraction of nuclei (first fraction to purify germline nuclei or second fraction to purify nuclei from any somatic tissue) as follows: Phycoerythrin-coupled anti-GFP antibody (Biolegend # 338003) was added to clarified nuclei in 7 to 8 ml of buffer A at 1:200 dilution. 280 units of murine RNAse inhibitor (M0314S) was added to protect RNA from being degraded. Nuclei were incubated in dark for 1 h or up to 16h at 4 C slowly rotating. Nuclei were clarified again after incubation (100 g for 6 min at 4 C) then pelleted (2000 g for 20 min at 4 C). Pelleted nuclei were then washed in 6 ml of buffer A and resuspended in 6 ml of buffer A + 1:500 of murine RNAse inhibitor and filtered on 30 µm mesh (CellTrics 04-0042-2316). Nuclei were stained with 0.025 µg/ml DAPI and their quality was assessed immediately before sorting by observation at 40X magnification. Nuclei sorting was performed using a Sony SH800Z sorter fitted with a 100 µm sorting chip and auto-calibrated. The nuclei were kept at 4 C during and after sorting. Background noise and debris were discarded by using the DAPI intensity signal as detection threshold. 2N single nuclei were gated using the DAPI signal and PE-positive nuclei were gated using PE-H / BSC-A signal. A recording speed > 15,000 nuclei per second ensured a sorting efficiency higher than 80 %, and nuclei were sorted in batches of one million and then processed for downstream applications immediately to limit the time they sit in buffer post-sorting. Nuclei were sorted into 15 ml Falcon tubes coated and filled with 500 µl of buffer A + 2 % RNAse-free BSA + 1:50 murine RNAse inhibitor (10 µl). Sorting purity was assessed before sorting large batches of nuclei and immediately after sorting for each batch, by microscopy and by recording sorted nuclei in a second pass in the sorter. A purity higher than 95 % was considered satisfactory. A batch of one million sorted nuclei was pelleted (2000 g for 20 min at 4 C) and resuspended in 10 µl of buffer A and topped up to 200 µl with 1X Tn5 Buffer (10mM Tris pH 8, 5mM MgCl2, 10% DMF). 47.5 µl of nuclei suspension were then aliquoted and 2.5 µl of Tn5 were added to the suspension. The mix was incubated for 30 min at 37 C while mixing at 400 rpm. Tagmented DNA was purified using a MinElute column (Qiagen) and converted into a library using the Nextera kit protocol. Typically, libraries were amplified using 12-16 PCR cycles. Libraries were then cleaned up using 0.6 volumes of beads. Beads bound by large fragments of DNA (> 700bp) were discarded and DNA recovered from the supernatant by adding 1.2 volumes of beads. DNA was eluted in 50 µl water and 0.9 volumes of beads were used to bind the library, leaving adaptor dimers in the supernatant. DNA was eluted in 20 µl water, quantified using a Qubit, and analyzed using an Agilent Tapestation. ATAC-seq libraries were made from two biological replicates for each tissue and sequenced in both single-end and paired-end modes.