GSM5452260: PAM-1 ChIRP-seq input; Mus musculus; OTHER
10 million cells were collected per ChIRP experiment for separated odd and even probe pools. Cell pellets were cross-linked with 1% Glutaraldehyde in 40mL PBS on rotator for 10 minutes at room temperature, followed by quenching the cross-linking reaction with 2mL 1.25M Glycine for 5 minutes and resuspend in 1mL chilled PBS. Cell pellets were collected at 2000RCF for 5 minutes at 4 °C, followed by removing PBS, snap frozen with liquid nitrogen and stored at -80°C. Cell pellets were lysed and sonicated using Covaris S220 sonicator, then aliquoted into two 1mL samples. Before ChIRP experiment, DNA were extracted for quality control with size ranging from 100-500bp. For ChIRP experiment, 10mL of lysate were saved for DNA input. 1mL of sonicated lysate was mixed with 2mL of hybridization buffer (750mM NaCl, 50mM Tris-HCl pH7, 1mM EDTA, 1% SDS, 15% Formamide, 1x protease inhibitor and 1x RNase inhibitor). 100pmol of odd and even ChIRP probes were added separately to the hybridization mixture and incubate at 37°C for 4 hours with rotation. After the hybridization was completed, 100uL of streptavidin magnetic C1 beads (Life Technologies, 65001) were washed thrice with hybridization buffer and added to each ChIRP reaction for extra 30 minutes incubation at 37°C with rotation. After the hybridization completed, 1mL of wash buffer (2X SSC, 0.5% SDS and 1x protase inhibitor) was used to wash the beads for 5 times using magnetic stand. Input control and PAM-1 bound DNA was eluted, followed by library construction. NEBNext Ultra II DNA Library Preparation kit for Illumina (NEB)