title: GSM6703653: DEtail-seq ssDNA with 3N in 3' end; synthetic construct; OTHER
library_construction_protocol: 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).