GSM2416921: ESd4_bc4_PAR_4C-seq_BioRep1; Mus musculus; OTHER
6-8 million cells were trypsinized and crosslinked in 40 mL of media with 2% formaldehyde for 10 minutes at room temperature. The formaldehyde was quenched by adding glycine to a final concentration of 125 mM; cells were then washed twice in 1X PBS, frozen in liquid nitrogen and stored at -80°C. To prepare 3C libraries, we used a method similar to the “in situ Hi-C” method (Nagano et al., 2015). Cells were lysed by resuspending them in 500 ml permabilization buffer (10 mM Tris-HCl pH=8.0, 10 mM NaCl, 0.2% Igepal CA-630) supplemented with 50 ml 100X EDTA-free protease inhibitor cocktail for 30 minutes on ice with occasional agitation to keep them from settling. Cells were spun at 1350g for 5 minutes at room temperature, then resupsended in 358 ml 1.25X DpnII buffer + 11 ml 10% SDS. Cells were rotated for 1 hour at 37°C, then 75 ml 10% Triton X-100 was added and cells were rotated at 37 °C for an additional hour to quench the SDS. To digest the chromatin, we added 5000 units of DpnII (NEB) and rotated the lysate at 37°C overnight. To perform in-situ ligation, we added 820 ml 10X T4 DNA Ligase Buffer (NEB), 82 ml 10 mg/ml BSA (NEB), 7.61 ml water and 50 ml T4 DNA Ligase (Enzymatics) and rotated for 4 hours at room temperature. To purify the DNA, 2 mg Proteinase K was added to the ligation reactions and left at 65°C overnight, then the DNA was cleaned by phenol:cholorform:isoamyl alcohol extraction followed by ethanol precipitation. We then treated the DNA with 40 ug RNaseA for 15 minutes at 37°C and performed a SPRI cleanup using a 1.5:1 bead:DNA ratio. We converted the 3C libraries into 4C libraries, using a protocol similar to the modified-4C protocol (Apostolou et al., 2013), but with additional modifications. First, we digested 3 mg of each 3C library with 4 units of FatI (NEB) for 4 hours at 55 degrees. We then dephosphorylated the ends of the FatI digested molecules by adding 20 units of Calf Intestinal Phosphotase (NEB) and heating at 37°C for 1 hour. The DNA was then purified using the PCR Purification Kit (Qiagen). To ligate the universal adaptor to the 3C library, we incubated the 3C library with 200 picomoles universal adaptor and 2 ml T4 DNA Ligase (Enzymatics) in 1X T4 DNA Ligase Buffer (NEB) in a volume of 50 ml for 1 hour at 16°C, then 2 hours at 25°C. We then purified the DNA and removed excess adaptor by adding 90 ml Ampure XP SPRI beads and performing a standard SPRI cleanup. Next, we used ligation-mediated PCR (LM-PCR) to selectively amplify molecules containing the viewpoint within the PAR region. We performed LM-PCR using 1.5 mM biotinylated viewpoint primer, 1.5 mM Illumina Index Primer in a 100 ml reaction with Phusion HF buffer as the master mix for 12 cycles. We then performed a SPRI cleanup with a 1:1 bead:PCR reaction ratio to remove unincorporated primers. To enrich for library molecules containing the viewpoint primer, we washed 10 ml MyOne C1 Streptavidin Beads (ThermoFisher) 3 times in 2X Binding and Wash buffer (10 mM Tris-HCl pH-8.0, 2M NaCl, 1 mM EDTA, 0.1% Tween-20), then incubated the washed beads with the purified PCR products for 15 minutes with rotation. We then washed twice with 1X Binding and Wash Buffer (10 mM Tris-HCl pH-8.0, 1M NaCl, 1 mM EDTA, 0.1% Tween-20), and once in TE+Tween (10 mM Tris-HCl pH-8.0 mM EDTA, 0.1% Tween-20). To incorporate full-length Illumina primer sequences, we added the beads to 100 ml PCR reactions, using 2 ml Illumina TruSeq Universal primer and 2 ml Illumina Index primer and Phusion HF master mix for 13 cycles. We then purified and size selected using a 0.85:1 bead:PCR reaction ratio. Library sizes were determined using the High Sensitivity Bioanalyzer (Agilent), and quantifications were determined using Library Quantification Kit (KAPA Biosystems). 2x76 bp paired-end reads were obtained using an Illumina MiSeq. 4C-PAR DpnIIview-point primer: /5Biosg/ccctacacgacgctcttccgatctNNNNCGAGAACGGGTGTCTCGGGGTT
GEO Accession
GSM2416921