Gentra puregene tissue kit

DNA was isolated from starved OP50 NGM plates with WT(drIs4) or mutant animals using the Qiagen Gentra Puregene Tissue Kit (Cat No 158667). The supplementary protocol for “Purification of archive-quality DNA from nematode suspensions using the Gentra Puregene Tissue Kit” available from Qiagen was used to isolate DNA. DNA samples were sequenced by BGI Americas (Cambridge, MA) with 20X coverage and paired-end reads using the Illumina HiSeq X Ten System.

Genomic DNA from frozen tissue was extracted using the Qiagen Gentra Puregene Tissue Kit (Qiagen). Sequencing libraries were prepped with the KAPA HyperPrep Kit (Roche) using 1 µg of DNA. DNA was sheared using a Covaris LE220 ultrasonicator targeting 200 bp, and sequencing adaptors added by ligation. Individually barcoded libraries were pooled 4-plex before capture. Libraries were hybridized to SeqCap EZ Choice probes of the 50 Mb Human UTR Design (Roche), and sequenced on a HiSeq 2500 (Illumina) using a PE100 in high-output mode. Image analysis and base calling were performed using Illumina’s Real Time Analysis v1.18.66.3 software, followed by demultiplexing of indexed reads and generation of FASTQ files, using Illumina’s bcl2fastq Conversion software v1.8.4.

Genomic DNA from mice exposed to DOX was extracted with the QIAGEN Gentra Puregene Tissue Kit (Qiagen Sciences) following the manufacturer’s instructions with minor modifications. In brief, frozen liver tissues (270–390 mg) were minced with a razor blade while on dry ice. The minced tissues were lysed with 3 mL cell lysis solution and incubated for 5 min on ice to allow for degradation. The tissue was then homogenized using a tissue homogenizer set at low-medium speed for no more than 1 min. Additional 3 mL of cell lysis solution were added and mixed by inverting 25 times. Next, 30 μl of Proteinase K (20 mg/mL) were added and tubes were mixed by inverting 25 times and incubated overnight in a shaker at room temperature. A total of 30 μl RNase A solution (4 mg/mL) was added to each lysate and mixed before incubation for 2 h in a shaker at room temperature. Then, 2 mL of protein precipitation solution were added and tubes were vortexed vigorously for 20 s prior to centrifugation (2500 x g for 15 min). Supernatants were added to cold IPA, and DNA was precipitated and washed as previously described, with the only difference being the DNA pellets were air-dried. The DNA pellets were stored at − 20 °C. The amounts described above were reduced by a factor of 4 when using 50 mg of liver tissue.