title: GSM4197395: Hypodermis_YA_RNA_pe_rep1; Caenorhabditis elegans; RNA-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. RNA was extracted from batches of one million sorted nuclei by pelleting nuclei (2000 g for 20 min at 4 C), washing them in 1 ml of buffer A, then adding 500 µl of Trizol to pelleted nuclei. 100 µl of Chloroform were added and samples were shaken vigorously for 15 s. Samples were then spun at 12,000 g for 15 min at 4 C and the aqueous phase was transferred into one volume of ice-cold Isopropanol. 0.5 µl of GlycoBlue (AM9515) was added and RNA was left to precipitate overnight at -20 C. Once precipitated, RNA was pelleted (12,000 g for 30 min at 4 C), washed in 1 ml of ice-cold 80% Ethanol and resuspended in 12 µl of RNAse-free water. Concentration was estimated using Qubit. A minimum of 20 ng of total nuclear RNA was used to make long nuclear RNA-seq libraries. Long nuclear RNA (>200 nt) was isolated using Zymo Clean and Concentrate columns (#R1013) and stranded libraries were prepared using the NEB Next Ultra Directional RNA Library Prep Kit (#E7420S) after removing rRNA using the Ribo-Zero rRNA removal kit (MRZH11124). Long nuclear RNA-seq libraries were made from two biological replicates for each tissue.