GSM5775963: Grem1 lineage traced cells - plate6; Mus musculus; RNA-Seq
Hind limbs were collected, and all muscles and ligaments removed. AC and GP from Grem1-TdT mice were excised under a dissection microscope and minced using a scalpel before being digested in 2.5mg/mL collagenase type II (#CLS-2, Worthington). Cleaned LepR-TdT whole bones were gently disrupted using a mortar and pestle, minced, and digested in 2.5mg/mL collagenase type I (#CLS-1, Worthington). The following antibodies were used: anti-CD45 (#103111, BioLegend), anti-Ter119 (#116211, BioLegend), anti-CD31 (#102509, BioLegend), and DAPI (#D9542, Sigma-Aldrich). LepR cells were sorted based on forward and side scatter, single cells, live cells (DAPI negative), trilineage negative expression (CD45-Ter119-CD31-) and TdTomato positive fluorescence. Grem1 cells were sorted based on forward and side scatter, single cells, live cells (DAPI negative), and TdTomato positive fluorescence. Individual cells were collected into single well in a 96-well plate with lysis buffer previously prepared by the Sulzberger Genome Center (Columbia University) and sequenced. Cells were randomly allocated into 96-well plates to avoid batch effects. Sequencing libraries were constructed using Nextera XT (Illumina) with modifications. A custom i5 primer was used (NexteraPCR) with 0.6ng input cDNA and 10 cycles of amplification was performed. Unique i7 indexes were used for each plate. After amplification, the library was purified with two rounds of AMPure XP beads, visualized on the TapeStation and quantified using the Qubit II fluorometer. Libraries were sequenced on an Illumina NextSeq 500 using the 75 cycle High Output kit (read lengths 26(R1) x 8(i) x 58(R2)). With each plate we targeted ~70M reads. Library pools were loaded at 1.8pM with 30% PhiX