title: GSM8394745: Fracture callus, CTL material, 7 d #2; Mus musculus; RNA-Seq
library_name: GSM8394745
library_construction_protocol: To obtain single-cell suspensions from calluses of tibiae, mice were euthanized, and calluses were minced using a scalpel in 100 U ml−1DNase I (Millipore Sigma, 10104159001), 1% bovine serum albumin (Sigma-Aldrich) and PBS for 20 min at 37 °C. The cell suspension was filtered using 40-μm cell strainers and centrifuged at 300 g for 5 min and treated with RBC lysis buffer (Millipore Sigma, 11814389001) for 10 min. Cells were stained with propidium iodide and viable cells were sorted using DiVa cell sorter (BD Biosciences) for scRNA-seq preparation. Cell suspensions harvested from calluses of tibiae were loaded onto a 10x Genomics Chromium Controller Single-Cell Instrument (10x Genomics) mixed with reverse transcription (RT) reagents, gel beads, and oil to generate single-cell gel bead-in emulsions (GEMs). GEM–RT was performed using an Eppendorf Mastercycler Pro using the following program: 53 °C for 45 min, 85 °C for 5 min. After RT, GEMs were broken, and the single-strand cDNA was purified using DynaBeads MyOne Silane Beads (Thermo Fisher Scientific). cDNA was then amplified using the Eppendorf Mastercycler Pro using the following program: 98 °C for 3 min, 11–13 cycles of 98 °C for 15 s, 67 °C for 20 s and 72 °C for 1 min, 72 °C for 1 min. The amplified cDNA product was then purified using the SPRIselect Reagent Kit (0.6 × SPRI; Beckman Coulter). Indexed sequencing libraries were constructed using the reagents in the Chromium Single-Cell Library Kit 3´ v2 using the following steps: 1) fragmentation, end repair and A-tailing, 2) SPRIselect cleanup, 3) adapter ligation, 4) post-ligation cleanup with SPRIselect, 5) sample index PCR, and 6) post-index PCR cleanup. Sequencing libraries were transferred to BGI and were loaded onto a MGISEQ-2000 platform (MGI Tech) for sequencing.