Rabbit_D10_micro-C
The regenerating ear pinna tissues from four rabbits at 10 dpi were collected and dissociated into single cells as described in the scRNA-seq. Live cells were doubly crosslinked using 1% formaldehyde (ThermoFisher, 28906) for 10 minutes and 3 mM DSG (ThermoFisher, 20593) for 45 minutes, respectively. The crosslinking reaction was quenched with 2 M Tris buffer (pH 7.5) and washed twice with PBS. The fixed cells were recounted and stored in small aliquots with 1-2 million cells per tube. The cross-linked cells were incubated on ice with 100 uL complete MB #1 buffer for 20 min. After collection by centrifugation at 10, 000 g for 5 min, cells were washed and resuspended with complete MB#1. The chromatin was fragmented with MNase (Worthington Biochem, LS004798) at a suitable concentration for 10 min at 37 on thermomixer C (Eppendorf) with 850 rpm, which aims to yield approximately 80% mono-nucleosomal fragments. The digestion was stopped by adding 0.5 M EGTA and incubation at 65 for 10 min. After fragmentation, the pellets were collected by centrifugation and washed with cold MB #2. For better efficiency of T4 DNA ligation, 50 L pre-mix (5 L 10x NEBuffer 2.1, 1 L 100 mM ATP, 2.5 L 100 mM DTT, 34 L H2O, 2.5 L 10 U/L T4 PNK) was added to the pellets and incubated for 15 min at 37 . Large Klenow fragment (NEB, M0210L) was added into the reaction with incubation for another 15 min to remove 3 overhangs and fill in 5 overhangs for blunt ends formation. The DNA overhangs were filled with biotinylated nucleotides by incubating with 25 L pre-mix buffer (5 L 1 mM biotin-dATP, 5 L 1 mM biotin-dCTP, 0.5 L 10 mM dTTP+dGTP, 2.5 L 10x T4 DNA ligase buffer, 0.125 L 20 mg/mL BSA, 11.875 L ddH2O) for 45 min at 25. The reaction was quenched by adding 500 mM EDTA to a final concentration of 30 mM with incubation for 20 min at 65 . The chromatin was collected by centrifugation (13, 000 g, 5 min, 4 ) and washed with cold MB #3. The chromatin pellet was resuspended in 250 L T4 reaction solution (25 L 10x T4 DNA ligase buffer w/ATP, 1.25 L 20 mg/mL BSA, 12.5 L 400 U/L T4 DNA ligase) and incubated for 2.5h at RT with slow rotation. After collection by centrifugation (16, 000 g, 5 min, 4 ), the pellet was resuspended in 100 L exonuclease reaction solution (10 L 10x NEBuffer #1, 5 L 100 U/L exonuclease , 85 L ddH2O) and incubated for 15 min at 37 with interval mixing. After proximity ligation, the chromatin solution with Proteinase K, 10% SDS, and RNase A was incubated at 65 overnight for reverse crosslinking and deproteination. The DNA was purified by phenol: chloroform: isoamyl alcohol extraction and ethanol precipitation. The 250-400 bp sized bands were cut and purified through 1.5% agarose gel electrophoresis and Zymoclean Gel DNA Recovery Kits. The ligation products were incubated with DynabeadsTM MyoneTM streptavidin C1 beads (Invitrogen, 65001), followed by end repair, adapter ligation, and library amplification using the VAHTS Universal Pro DNA Library Prep Kit for Illumina (Vazyme, ND608-02). The 150-bp pair-end sequencing was performed for all the libraries. 【NOTE】This raw data is too large to download using prefetch with default parameters. Please using this command : 'prefetch --type any SRR329xxxxx --max-size 60000000' to download them. The SRA run is not empty!!!!!!!! Files over 100G have been splited into two files(part1, part2) for uploading. Please merge them first before running downstream pipelines.