G4P-ChIP library construction

Approximately 0.5–1 × 10⁸ transiently or stably transfected cells expressing G4P were crosslinked with 1% formaldehyde for 20 min at room temperature. Fixation was quenched by 0.125 M glycine for 15 min. The fixed cells were washed twice with PBS, suspended in NP-40 buffer (10 mM Tris–HCl pH 7.4, 150 mM NaCl, 0.5% NP-40 and 2 mM AEBSF) and incubated on ice for 10 min. After centrifugation at 800 × g for 5 min, the cell pellet was resuspended in a CHAPS buffer (20 mM Tris-HCl pH 7.4, 0.5 mM EGTA, 50 mM NaCl, 0.5% CHAPS, 10% glycerol and 2 mM AEBSF). The suspension was incubated on ice for 30 min and centrifuged at 800 × g for 5 min. The pellet was resuspended in 1 ml 1× dsDNase digestion buffer supplied with 50 μl dsDNase (Invitrogen, EN0771) and incubated at 37°C for 20 min with constant agitation. A final concentration of 20 mM EDTA was added to terminate the reaction. The samples were pelleted by centrifugation at 15 000 × g at 4°C and the resulting supernatant was collected and incubated on ice. The pellet was resuspended in 500 μl wash buffer (150 mM NaCl, 10 mM Tris–HCl, pH 7.4, 0.1 mM EDTA, 0.5% Triton X-100) and sonicated for 30–60 s in an ice-cold water bath. After centrifugation at 15 000 × g for 5 min, the supernatant of chromatin fragment was collected and combined with the supernatant from the previous step.

For library preparation, 50 μl of anti-FLAG M2 magnetic beads (Sigma-Aldrich) were washed with washing buffer (10 mM Tris–HCl, pH 8.0, 150 mM NaCl and 0.5% Triton X-100) and blocked in the same buffer containing 75 μg/ml single-stranded sperm DNA and 1 mg/ml BSA. 1% chromatin fragment was saved as input and the remaining was incubated with blocked anti-FLAG magnetic beads in rotation at 4°C for 3 h. The beads were sequentially washed ten times with washing buffer and transferred to new tubes three times. The chromatin was eluted with 300 μg/ml 3xFLAG peptide (Sigma-Aldrich) at 4°C for 1 h. The eluted chromatin and the input samples were incubated with proteinase K at 65°C overnight. After sequential RNase A and proteinase K digestion, DNA fragment was cleaned by extraction with phenol:chloroform:isoamyl alcohol, followed by ethanol precipitation. Libraries were constructed from the recovered DNA fragment using the NEBNext Ultra II DNA LibraryPrep Kit from Illumina (NEB) according to the manufacturer's instructions. The next-generation sequencing was performed with Illumina HiSeq X Ten by Genewiz (Suzhou, China).