GSM9415008: 0914_HP1BP3_CUTTAG; Mus musculus; OTHER
Samples were minced into pieces <5 mm and then homogenized using a glass dounce tissue grinder in 2 mL of Nuclei EZ lysis buffer on ice. After two incubations on ice (with 4 mL of lysis buffer and 5 min each time), the homogenate was filtered through a 70 mm strainer, and then we used Debris Removal Solution to perform density gradient centrifugation to clean the nuclear suspension according to the manufacturer's protocol. Isolated nuclei were resuspended and washed with nuclei suspension buffer (NSB, consisting of 13 mL PBS, 0.1% mL BSA and 0.4 U/mL Ambion RNase inhibitor and filtered through a 35 mm cell strainer.Nuclei were counted using a hemocytometer and diluted to 1000 nuclei/mL for optimal 10X loading. For library amplification in PCR tube format, 21 µL DNA was combined with 2 µL of universal i5 and uniquely barcoded i7 primer where a different barcode was used for each sample that was intended to be pooled together. 25 µL NEBNext HiFi 2× PCR Master mix was added, and then the sample was gently mixed through and spun down. The sample was placed in a Thermocycler with heated lid following these conditions: 72 °C for 3 min (gap filling); 95 °C for 3 min; 10 cycles of 98 °C for 10 s and 60 °C for 5 s; final extension at 72 °C for 1 min; and hold at 4 °C. Following PCR, bead cleanup was conducted by the addition of 1.1x Ampure XP beads. Library and beads were mixed thoroughly, then spun down and incubated at RT for 10–15 min. Beads were gently washed twice with freshly prepared 80% ethanol using a magnetic rack, then the library was eluted with 20 µL 10 mM Tris-HCl pH 8.0 at RT.(Illumina novaseq6000，PE150)