title: GSM7225276: Esco2fl/fl;Prrx1-CreTg/0, mutant, E9.5, 2; Mus musculus; RNA-Seq
library_construction_protocol: Dissections were performed under a stereomicroscope in multiple changes of cold phosphate-buffered saline (PBS). Limb buds were digested at 37 °C in 0.5 mL of 0.25% trypsin/EDTA (Invitrogen, 25200-072) for 5 minutes at 37°C with trituration by pipetting at 2.5 and 5 minutes. Trypsinization was stopped by adding 0.5 mL of cell culture medium DMEM (CO & CAT #) with 10% FBS (CO & CAT #). Cell suspensions were centrifuged at 300g and 4 °C for 3 minutes. Supernatant was removed, and cells were resuspended in 0.5 mL of pre-chilled (4 °C) 10% DMSO in FBS. Cell suspensions were then transferred into cryovials, placed into a pre-chilled freezing container (CO & CAT #) at -80°C overnight, and transferred to -150°C for long term storage the following day. Once sufficient samples were generated, cryovials were thawed at 37 °C and transferred into a 15 mL centrifuge tube. 1 mL of pre-warmed (37 °C) cell culture medium was added dropwise with gentle tube shaking, followed by an additional 2 mL of medium. Three samples were then pooled into one 15 mL centrifuge tube and mixed well prior pelleting at 300 g for 3 minutes. The media was then aspirated slowly leaving approximately 50 µl of supernatant and cells were resuspended in 1 mL of freshly made 0.04% BSA in PBS. Cell suspensions were then strained through a 40 μm filter (Falcon, 352340), pelleted at 300g for 3 minutes, aspirated to approximately 50 µl of supernatant, and resuspended in 50 uL of 0.04% BSA in PBS. scRNA-seq 3' expression libraries were prepared on a Chromium instrument (10X Genomics, model  GCG-SR-1) using the Chromium Single Cell Gene Expression kit (version 3).