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Protocol publication

[…] All experiments were performed following protocols approved by the Harvard University IACUC committee. Timed matings were established between FVB mice, and pregnant females were euthanized at E15.5 in order to acquire embryonic long bone growth plates. Embryos were dissected in PBS1X on ice under a dissection scope and the proximal and distal growth zones of the right and left femur were stripped clear of soft tissues. Each proximal or distal cartilaginous end was then micro-dissected from the bony diaphysis () and separately pooled from a single litter, consisting on average of eight animals. Two biological replicates were collected in line with previous ATAC-seq studies (). The samples were collected in micro-centrifuge tubes containing 200 ul 5% FBS/DMEM. To generate a single-cell chondrocyte suspension, each pooled sample was then subjected to 1% Collagenase II (VWR 80056–222, Radnor, Pennsylavania) digestion for 2 hr at 37°C rocking, mixing every 30 min. After placing on ice, samples were filtered using a micro-centrifuge filter set-up by gently mashing the residual tissues through the filter followed by rinsing with 5% FBS/DMEM. Samples were then spun down at 500 g at 4°C for 5 min. We next performed cell counting methods using trypan blue and a hemocytometer and performed subsequent ATAC-seq steps on those samples that had cell death rates well below 25%. On average we acquired 500,000–1,000,000 living cells per harvest. Next, cells were re-suspended in concentrations of 50,000 cells in 1x PBS. Cell samples then subjected to the ATAC-seq protocol as described in , modifying the protocol by using 2 µl of transposase per reaction. The transposase reaction product was then purified using the Omega MicroElute DNA Clean Up Kit following manufacturers protocols, eluted in 10 µl of warmed ddH20, and stored at −20°C.Next, samples were subjected to PCR amplification and barcoding following . All primers used in this step are listed in . Ten microliters of transposed DNA were then placed in a reaction containing NEBNext High-Fidelity PCR Master Mix, ddH20, and primers. After amplification, samples were transferred to micro-centrifuge tubes and subjected to the OMEGA Bead Purification Protocol following manufacturers protocol. The samples were eluted in 30 µl of TE, run on a nanodrop, diluted to 5 ng/µl and run on a bioanalyzer. Prior to sequencing sample concentrations were determined using the KAPA Library Quantification Complete Kit (KK4824). Samples were then sent out to the Harvard University Bauer Core Facility for sequencing on one lane of the Illumina NextSeq 500 (see ).Sequencing yielded approximately 400 million reads per lane and an average of 100 million per sample. Quality control statistics are presented in . Sequenced reads were next aligned to the mouse reference mm10 genome assembly using Bowtie2 (). Duplicated reads were removed and significant peaks determined by using MACS2 software (version 2.1.0) (). Peaks were assessed for reproducibility between biological replicates using the IDR statistical test () at various statistical cut-offs, with an IDR threshold of <0.05 selected to define reproducible peaks for all subsequent analyses. Datasets for other IDR score thresholds are available upon request and when used in all analyses show similar statistical enrichments. Intersections between proximal and distal femur peaks were done with IDR-filtered narrow peak files, with the proximal/distal-common set generated by merging overlapping peaks. Annotations of the narrow peak files generated on all datasets were made using HOMER ( sequencing fastq files and processed peak bed files have been deposited on NCBI GEO (GSE100585). […]

Pipeline specifications

Software tools Bowtie2, MACS, HOMER
Application ATAC-seq analysis
Organisms Mus musculus, Homo sapiens