GSM5850926: ATAC-seq Col10a1-mCherry Proximal Femur E15.5; Mus musculus; ATAC-seq
The samples were collected in microcentrifuge tubes containing 200ul 5% FBS/DMEM. To generate a single cell chondrocyte suspension, each pooled sample was then subjected to 1% Collagenase II (VWR 80056-222) digestion for 2hrs at 37C rocking, mixing every 30 minutes. 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 500g at 4C for 5 min. We next performed cell counting methods using trypan blue and a hemacytometer 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 resuspended in concentrations of 50,000 cells in 1x PBS.  Cell samples then subjected to the ATAC-seq protocol as described in Buenrostro et al., 2015, modifying the protocol by using 2 ul 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 ul of warmed ddH20, and stored at -20C. Samples were subjected to PCR amplification and barcoding following Buenrostro et al., 2015. 10 ul of transposed DNA was 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 ul of TE, run on a nanodrop, diluted to 5 ng/ul 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