Instrument: Illumina NovaSeq 6000
Strategy: OTHER
Source: GENOMIC
Selection: other
Layout: PAIRED
Construction protocol: Samples collected were lysed in genome extraction buffer (200 mM NaCl, 10 mM Tris-HCl pH 8.0, 10 mM EDTA, 0.5% SDS, 200 μg /ml freshly added Proteinase K) at 37°C for 8~12 h. Then, 1/4 volume of 5 M potassium acetate solution (pH 5.2) was added to the lysate and mixed gently, followed by incubation on ice for 20 min. Following extraction with equal volume of phenol: chloroform: isoamyl alcohol (25:24:1, pH 7.8) and chloroform sequentially with phase-lock tubes. The supernatant containing genomic DNA was precipitated by addition of equal volume of isopropanol and 1 μl GlycoBlue (Thermo Fisher) for 30 min at -20°C. The precipitate was washed by 70% ethanol for once and then air-dried. Genomic DNA was digested with mung bean nuclease (TaKaRa) at 37°C for 2 h and purified by the phenol-chloroform method followed by resuspending the pellet in 130 μl TE buffer. Digested gDNA was sonicated to a peak fragment size of 250 bp, performed on a S220 Focused-ultrasonicator with 10% Duty Factor, 200 cycles/burst, 175 peak incident power and for 90 sec per tube. For inter-group relative quantification, gDNA from Arabidopsis (7-day-old Col-0 seedlings) was used as the spike-in sample to normalize total R-loop levels between animal samples. Sonicated spike-in gDNA to a peak fragment size of 250 bp as described above, and then added ~0.1 ng sonicated spike-in gDNA to sonicated target gDNA samples. 80% of the mixed sample was used for the first adapter ligation of ULI-ssDRIP-seq workflow, and the rest 20% part was used for input-library preparation by using Accel-NGS 1S Plus DNA Library Kit following the manual. For the first adapter ligation (pre-ligation), 53 μl sonicated gDNA was added to Adaptase reaction mix (for each sample: 8 μl buffer G1, 8 μl reagent G2, 5 μl reagent G3, 2 μl enzyme G4, 2 μl enzyme G5, and 2 μl enzyme G6, from Accel-NGS 1S Plus DNA Library Kit), incubated at 37°C for 1 hour. Then DRIP was performed as described previously 5. Prepared 174 ul pre-mixed extension reaction mix following the extension part of manual (Accel-NGS 1S Plus DNA Library Kit) and mixed with beads/antibody complexes, followed by 1 cycle PCR (98°C 90 sec, 63°C 30 sec, 68°C 5 min). Purified DNA by adding 1.2 volumes of SPRIselect beads. Ligated the second truncated adapter to the 5' ends at 25°C for 1 hour following the ligation part of manual (Accel-NGS 1S Plus DNA Library Kit). Purified DNA again by 1 volume SPRIselect beads and eluted with 21 ul Low EDTA buffer. 1 ul purified DNA was used for qPCR by using indexing primers to address cycle numbers of indexing PCR. The cycle numbers of indexing PCR was equal to the Ct value. An indexing PCR step was performed following the manual of Accel-NGS® 1S Plus DNA Library Kit.