title: GSM8163698: Calvarial bone-PLD14, replicate 1, scRNAseq; Mus musculus; RNA-Seq
library_construction_protocol: Skulls with calvarial lesions (diameter: 1 mm) at PLD14 with their corresponding age-matched controls were harvested, cleaned from attached surrounding tissue and collected in digestion enzyme solution (Collagenase type I and IV, 2 mg/ml). Next, bones were cut into small pieces. Samples were digested for 30 min at 37 °C under gentle agitation. Digested samples were transferred to 70 µm strainers in 50 ml tubes to obtain a single–cell suspension, which was resuspended in blocking solution (1% BSA, 1mM EDTA in PBS without Ca2+/Mg2+), centrifuged at 300×g for 5 mins, washed 2–3 times with ice-cold blocking solution, and filtered through 50 µm strainers. Pellets were resuspended in respective volume of blocking solution. Single cell suspensions were subjected to lineage depletion using lineage cell depletion kit (MACS, cat#130-090-858) following the manufacturer's instructions. Next, lineage negative cells were depleted by CD45 and CD117 using microbeads (MACS, cat#130-052-301 and cat#130-091-224) from lineage negative (Lin-) bone cells to enrich bone stromal cells. Single cell suspensions were processed with BD Rhapsody single cell RNA-seq. scRNA-seq libraries were evaluated and quantified by Agilent Bioanalyzer using High sensitivity DNA kit (cat#5067-4626) and Qubit (ThermoFisher Scientific, Cat# Q32851). Individual libraries were diluted to 4 nM and pooled for sequencing. Pooled libraries were sequenced by using High Output kit (150 cycle) (Illumina cat#TG-160-2002) with a NextSeq500 sequencer (Illumina).