title: GSM4668507: mmPac1; Mus musculus; OTHER
library_construction_protocol: Testes from adult C57BL/6 mice were removed and decapsulated. The seminiferous tubules were tore into small pieces and incubated in 8 ml PBS containing 1 mg/ml collagenase (Sigma, C5138, St. Louis, MO, USA) and 1 mg/ml hyaluronidase (Sigma, H3506, St. Louis, MO) at 37°C for 6 min with gentle shaking. After pipetting, the dispersed seminiferous tubules and cells were incubated at 37°C for 5 min with gentle shaking. Then, the cells were collected by centrifugation at 200 × g for 5 min at 4°C, washed once with PBS, resuspended in 15 ml PBS containing 0.25% Trypsin and 1 mg/ml DNase I, and incubated at 37°C for 5 min with gentle shaking. Thereafter, the cells were collected and washed with PBS containing 0.5% BSA. After filtration through a 40 μm Nylon Cell Strainer, the cells were separated by sedimentation velocity at unit gravity at 4°C, using a 2–4% BSA gradient in PBS. The cell fractions were bottom-loaded in a volume of 300 ml. An aliquot of each fraction was examined by light microscope to assess cellular purity and cell type identity. Each cell pellet was resuspended in 150 µl 50 mM EDTA pH 8.0 and allowed to thaw at 40°C. 498 µl LMP agarose (2% in 50 mM EDTA pH 8.0) was mixed with 102 µl Solution 1 (1 M sorbitol, 0.1 M sodium citrate, 60 mM EDTA pH 7.0, 5% β-mercaptoethanol and 1 mg/ml zymolyase 100T) and the mix was pre-warmed at 40°C before adding to the cells. The resuspended cells were pipetted into plug molds and chilled at 4°C for 30 min. Once solidified, the agarose plugs were cut into small pieces (one agarose plug to 16~24 pieces), and expressed into 1 ml of Solution 2 (0.45 M EDTA pH 8.0, 0.01 M Tris-HCl pH 7.5, 7.5% β-mercaptoethanol, 10 µg/ml RNase A) and incubated at 37°C for overnight. Plugs were quickly washed three times with 1 ml TE buffer, and then washed five times with TE buffer incubated at 37°C for 6min (total 30min). Then plugs were expressed into 1ml Solution 3 (0.25 M EDTA pH 8.0, 0.01 M Tris-HCl pH 7.5, 1% SDS, 1 mg/ml proteinase K) and incubated at 54°C overnight. Plugs were finally washed six times with TE buffer and stored in plug storage solution (0.05 M EDTA pH 8.0, 50% glycerol) at -20°C until all plugs were ready to be processed for library preparation.  The plugs were transferred to a PCR tube, and washed by 30 µl 1X Adaptase buffer (Swift, Accel-NGS, 10096) twice at room temperature, 10 min each time. Add 47 µl 1X Adaptase buffer and 3 µl Adaptase (Swift, Accel-NGS, 10096) to PCR tube, incubated with plugs at 37°C for 2 h. Removed buffer, and deactivated Adaptase in plugs by 98°C for 2 min. Purified DNA from plugs by Gel Extraction Kit (Qiagen, 28606). Sonicated DNA to 150~250 bp fragments by using Focused-ultrasonicator (Covaris, S220). The rest steps were following the manual of Accel-NGS 1S Plus DNA Library Kit (Swift, Accel-NGS, 10096). Briefly, denatured DNA fragments to ssDNA, and extended it to dsDNA. After purification, 2rd adaptor was ligated. After indexing PCR and purification, preparation of DEtail-seq library was finished. The DEtail-seq library was sequenced by NovaSeq 6000 (Illumina).
attributes.tag: GEO Accession
attributes.value: GSM4668507