GSM6637218: ATH_ChIP_rep1; Arabidopsis thaliana; OTHER
For ssDRIP-seq, the chloroplasts were extracted by grounding plant leaves in 20 ml ice-cold CIB buffer. The homogenate was filtrated through two layers of Miracloth and centrifuged at 200 g/4°C for 3 min. Then the supernatant was transferred to new 50 ml tubes and centrifuged for 10 min at 1200 g/4°C. The chloroplasts were then suspended with cold CIB buffer and was ready for use. The chloroplasts were lysed in chloroplast DNA extraction buffer (CIB with 1% SDS and proteinase K) at 37°C overnight with shaking, and then SDS was removed by adding 20 µM KAc. Chloroplast DNA was purified by phenol/chloroform/isoamyl alcohol (25:24:1, v/v/v) and precipitated with an equal volume of isopropyl alcohol at -20°C overnight. The chloroplast DNA was fragmented with 5 U of DdeI (NEB, R0175V), MseI (NEB, R0525S), RsaI (NEB, R0167V), and AluI (NEB, R0137V) at 37°C for 12 h and then purified. 2 µg of purified fragment DNA with or without RNase H treatment was incubated with 10 µg of S9.6 antibody overnight at 4°C. Samples were further incubated with 50 µl Protein G beads (Invitrogen, 10004D) for 4 h at 4°C. The immunoprecipitated DNA was purified as mentioned above. For ATH ChIP-seq, the chloroplasts were cross-linked with 1% formaldehyde for 10 min, and the cross-linking reaction was stopped by adding 150 µl 1 M glycine and incubating for 10 min. Then the cross-linked chloroplasts were washed twice with CIB and lysed in lysis buffer (50 mM Tris-HCl (pH 7.6), 0.15 M NaCl, 1 mM EDTA (pH 8.0), 1% Triton X-100, 0.1% SDS, 0.1% sodium deoxycholate). Chloroplast DNA was sheared with sonication into fragments of ∼500bp. The supernatant was incubated with an anti-GFP antibody (abcam, ab290) overnight at 4°C. For DEtail-seq, the chloroplasts were embedded in low-melting-point agarose and lysed in lysis buffer as described above. Agarose plugs were then washed in 1x TE buffer 4 times at 37°C, with the first two washes containing 1 mM PMSF. Then, agarose plugs were cut into small pieces and lysed in 10 μg/ml RNase A at 37°C overnight. The lysed agarose pieces were then washed in 1x TE buffer 5 times at 37°C, followed by two washes with 300 μl 1x CutSmart (NEB, B7204S) at 37°C. The agarose pieces were then incubated with 3 μl I-CeuI (NEB, R0699S) in 150 μl 1x CutSmart Buffer at 37°C for 12 h and washed 3 times with 1x TE buffer. Then the T7 ligation process was conducted by adding 65 μl (T7 Buffer 8 μl, T7 Adapter 5 μl, T7 Enzyme Mix Ⅱ 6 μl, Low-EDTA TE 46 μl) T7 Tailing & Ligation solution (ABclonal, RK20228) and incubating at 37°C for 12 h. The DNA in agarose was purified using a DNA gel purification kit (Magen, D2111-02), and fragmented to ~250 bp using a focused ultrasonicator (Covaris, S220). The library preparation steps were conducted according to the manual (ABclonal, RK20228). Briefly, the first adapter was ligated to the 3' end of the ssDNA using Adaptase via a highly efficient, proprietary reaction that only tails 3' ends of ssDNA and ligates the first truncated adapter to 3' ends simultaneously. This method avoids the bias inherent in random primer-based methods, as it ligates adapters in a sequence-independent manner. The extension step was performed using the primer paired to the first adapter, followed by a ligation reaction to add the second truncated adapter to the 5' ends. An indexing PCR step was performed to add the indexed sequence, and the library was amplified.