title: GSM9167611: Young, d7, rep1, contralateral, HTO; Mus musculus; OTHER
library_name: GSM9167611
library_construction_protocol: The bone marrow was flushed from murine femora using a syringe with PBS containing 2% FCS and passed through a 70um cell strainer. Red blood cells were lysed using ACK lysis buffer for 1 min at RT, followed by washing with PBS + 2% FCS. Cortical bone fragments were subjected to digestion buffer (HBSS with 2 mg/mL collagenase IV and 1 mg/mL DNase II) at 37C for 30 min with gentle shaking. Following digestion, cells were passed through a 70um strainer, washed and resuspended in PBS + 0.04% BSA. Viable single cells were counted using trypan blue and used for downstream single-cell libraries. Single-cell suspensions from bone marrow and cortical bone were processed using 10x Genomics Chromium Single Cell 3' v3 chemistry (10x Genomics) according to the manufacturer's protocol. Cells were first labeled with TotalSeq-B hashtag oligonucleotide (HTO) antibodies (BioLegend) for multiplexing. After washing to remove unbound antibodies, labeled cells were loaded into the Chromium Controller for droplet encapsulation. Libraries for both gene expression (GEX) and HTO were generated from the same Gel Bead-in Emulsion (GEM) reaction. Following reverse transcription, GEMs were broken and cDNA was purified. Separate amplification and indexing steps were performed for GEX and HTO libraries, which were then pooled and sequenced according to the 10x Genomics recommendations.