GSM4227225: E13.5; Mus musculus; RNA-Seq
Murine hindlimbs were manually dissected at 4 time points during embryonic joint development, including points marking formation of the limb bud (E11.5), formation of cartilaginous condensations (E13.5), joint cavitation (E15.5), and joint morphogenesis (E18.5). Embryonic pups (7-9/litter) were removed by caesarean section from a timed-pregnant female C57BL/6 mouse per time point (The Jackson Laboratory, Bar Harbor, ME) and euthanized. Hindlimbs from 6-7 pups/litter were digested with collagenase (Type II, Worthington-biochem, Lakewood, NJ) and pronase (EMD Millipore, Billerica, MA) in 15-minute increments with agitation at 37°C for up to one hour. Every 15 minutes, mechanical dissociation was applied by pipetting up and down. Following digestion, cells from each litter (time point) were pooled, strained through a 100um cell strainer, pelleted, resuspended at 1 x 10^6 cells per 500ml in freeze media (80% FBS, 10% DMEM, 10% DMSO), and stored in liquid nitrogen until single cell capture. Cells in freeze media were rapidly thawed in a 37°C water bath, and resuspended in 1X PBS and 0.04% bovine serum albumin. Dead cells were removed using magnetic bead-bound antibodies against apoptotic and necrotic cells (Dead cell removal kit, Miltenyi Biotec) and cells were washed twice. Single cell capture was performed with the 10x Chromium Controller (10x Genomics, Pleasanton, CA). Briefly, single cell suspensions were brought to a concentration of 1,000 cells/ml and approximately 6,000 cells per time point (4 samples total) were loaded into the 8-channel microfluidic chip that encapsulates thousands of cells in gel beads in emulsion (GEMs). Cell capture, cDNA generation, and library preparation were performed using Chromium Single Cell 3' v2 Reagent kit, following the manufacturer's instructions. To test the reliability of single-cell capture, a 1:1 mixture of E18.5 mouse hindlimb cells and human 293T cells was sequenced and the multiplet rate was determined by the fraction of mouse reads in human barcodes and vice versa. Single cell RNA-seq libraries were pooled and run on the Illumina NovaSeq S1 flow cell with 26x98bp reads to generate ~500 million reads per library (time point).
GEO Accession
GSM4227225