GSM3504364
GSM3504364: S2-RNAPII-ChIA-PET (RNaseA - control); Drosophila melanogaster; ChIA-PET
Chromatin was obtained and subjected to fragmentation with an average length of 500 bp by sonication. The anti-RNAPII monoclonal antibody 8WG16 (Biolegend, 920102) was used to enrich RNAPII-bound chromatin fragments, respectively. The DNA fragments in the streptavidin beads-bound chromatin for RChIA-PET or antibody beads-bound chromatin for RNAPII RChIA-PET were end-repaired using T4 polymerase (Promega) in mixture (70 µl 10× T4 polymerase buffer, 7 µl 10mM dNTP, 3.5 µl RI, 612.3 µl Nuclease-free water, with 7.2 µl of T4 polymerase), washed with RChIA-PET Wash Buffer (10 mM Tris.HCl pH 7.0, 10 mM Tris.HCl pH 8.0, 10 mM EDTA, 50 mM NaCl) three times. The RNA fragments were linked to RNA linker for first-strand cDNA synthesis using the Superscript III First Strand Synthesis System (Thermo Fisher Scientific). In more details, 1 µg of biotinylated and barcoded RNA linker R1 (tube 1) and R2 (tube 2) containing a flanking MmeI site were added to two tubes containing cDNA synthesis mixture (20 µl RNA liker, 20 µl dNTP, 80 µl 5× RT buffer, 20 µl 0.1 M DTT, 20 µl RI, 220 µl Nuclease-free water) and incubated at 65 °C for 5 min (Alternative, incubated at 42 °C for 5 min when performing RNAPII RChIA-PET), then placed on ice for at least 1 min, and added 20 µl superScriptIII RT, and incubated for 10 min at 25 °C on Intelli-Mixer (Program F1, 10rpm), followed by 30 min at 50 °C (Alternative, 1 hour at 37 °C when performing RNAPII RChIA-PET). The RNA linker ligated chromatin was washed with Wash Buffer three times, then incubated with 1 µg of DNA linker D1 (tube 1) and D2 (tube 2) respectively in the ligation mixture (15 µl DNA linker, 140 µl 5× T4 DNA ligase buffer with PEG, 3.5 µl RI, 536.5 µl Nuclease free water) by using 5 µl of T4 DNA ligase at 16 °C for overnight. The linker-added DNA fragments were washed with Wash Buffer three times and then phosphorylated with 14 µl of T4 polynucleotide kinase (NEB) in PNK master mix buffers (70 µl 10× T4 DNA ligase buffer, 3.5 µl RI in 612.5 µl Nuclease-free water). The samples from two tubes were washed with Wash Buffer three times and then were mixed and followed by proximity ligation with 34 µl of T4 DNA ligase in reaction buffer (1,000 µl 10× T4 DNA ligase buffer, 50 µl RI, 8,916 µl Nuclease-free water) overnight at 16 °C. After washing with Wash Buffer three times, the chromatin DNA fragments with linkers were subjected to second-strand cDNA synthesis with a Superscript Double-stranded cDNA Synthesis Kit (Thermo Fisher Scientific) and chromatin mixing with second-strand cDNA mixture (333 µl Nuclease-free water, 90 µl 5× second-strand reaction buffer, 9 µl 10 mM dNTP mix, 3 µl E.coli DNA ligase, 12 µl E.coli DNA Polymerase I, 3 µl E.coli RNase H) were incubated at 16 °C for 2 hours; then 6 µl of T4 DNA polymerase were added and continued to be incubated at 16 °C for 5 min. Crosslinks in the DNA/RNA/protein complexes were then reversed by incubation at 65 °C overnight with 0.5% SDS (Thermo Fisher Scientific) and proteinase K (Thermo Fisher Scientific), and the cDNA-DNA fragments were purified by phenol/chloroform isopropanol precipitation. The purified cDNA-DNA was then digested by 1 µl of MmeI (NEB) in buffer (5 µl 10× NE Buffer 4, 5 µl Half linker non-Biotinylated to quench excess MmeI, 5 µl 10× SAM) for 2 hours at 37 °C to release the [cDNA tag]-[RNA linker]-[DNA linker]-[DNA tag] Paired-End-Tag structure. The biotinylated PETs were then immobilized on streptavidin-conjugated magnetic Dynabeads (Thermo Fisher Scientific, 11206D-10ml) in 50 µl of 2× B&W buffer (10 mM Tris-HCl pH 7.5, 1mM EDTA, 1 M NaCl), and rotated at room temperature for 45 min. The ends of each PET structure were then ligated to a sequencing adaptor by 1 µl of T4 DNA ligase (Thermo Fisher Scientific, EL0013) in Adaptor ligation buffer (4 µl Adaptor A, 4 µl Adaptor B, 5 µl 10× T4 DNA ligase buffer, 36 µl Nuclease-free water) at 16 °C for overnight with rotation. Then the beads were washed three times with 1× B&W buffer (5 mM Tris-HCl pH 7.5, 0.5 mM EDTA, 1 M NaCl). Nick translation was performed by adding 4 µl of E.coli DNA polymerase I solution mixture (38.5 µl Nuclease free water, 10× NEBuffer 2, 2.5 µl 10 mM dNTP) and incubated at room temperature for 2 hours with rotation on the Intelli-Mixer (F8, 30rpm, U = 50, u = 60). Then 10-16 rounds of PCR reactions were performed to amplify the PETs, and the resultant RChIA-PET libraries were subjected for sequencing by MiSeq with paired-end reads of 36 bp.
GEO
GSM3504364
GEO Accession
GSM3504364