title: GSM5105050: Matrigel 3D cultured non-limb fibroblast control for 4 d (mm10_hrepr_3); Mus musculus; RNA-Seq
library_construction_protocol: For sorting reprogrammed Prx1-GFP or Prx1-tdTomato cells from embryos, a FACS sorter Astrios (Beckman Coulter) or On-chip Sort HSG (On-chip Biotechnologies) was used. After washing with PBS, the cells cultured in the HA-gels or on Matrigel were incubated in TryPLE Express (gibco) for 30 min at 37℃. The cell suspension was pipetted with cut P1000 pipette tips every 10 min, to completely dissociate the cell clusters. The suspension was filtrated by 100 μm Cell strainers (Falcon) and 40 μm Cell strainers (VWR), and cells were pelleted by centrifugation (400 x g for 5 min). The pellets were dissociated by DRAQ5/DAPI in 0.1% BSA/PBS and incubated for 5 min before the sorting. DRAQ5-positive, DAPI-negative cells were sorted for cells on reprogramming at day 2, 4, 8. For HA-gel reprogrammed cells at day 14, additional gating on GFP channel derived GFP-positive and GFP-negative samples. For Matrigel-derived day 14 reprogrammed cells for PZL- as well as PZLL- factors, only GFP-positive cells were collected. DRAQ5-positive, DAPI-negative, Matrigel-derived day 8 cultured E9.5 and E10.5 limb progenitors were collected. The E9.5 cultured limb progenitors were subject to 4-OHT, such that large fraction were tdtomato-positive, but the cells were collected regardless of tdTomato-positivity. DRAQ5-positive, DAPI-negative, tdTomato-positive cells were sorted for the limb mesenchyme cells for E10.5, E11.5 as well as E12.5 cells. For E9.5 limb progenitors, samples were collected without tdTomato gating to maximize yield. InDrop cell capture was processed in Harvard Medical School Single Cell Core. All libraries included about 10-15% of Non-limb fibroblasts to mitigate batch effect. Cells were captured by the Chromium Controller (10X genomics, Biopolymer facility at Harvard Medical School). single cell RNA libraries were prepared for sequencing using 10X Genomics Chromium V3 protocols and inDrops libraries were prepared by the Harvard Single Cell Core according to the published protocol scRNA-seq