Single-cell transcriptional landscape of human embryonic limb development
Single-cell transcriptional landscape of human embryonic limb development
The human embryonic samples were collected from medical abortions. The samples were stored in hibernate E medium (Thermo Fisher A1370601) and transported to the research lab for dissection. The whole limb bud or upper part of the lower limbs were then dissected are stored in ice-cold PBS before single-cell isolation. Samples were chopped into a mash using a scalpel and incubated in 5ml of 2.5mg/ml liberase at 37 degrees for 30-40 min till fully digestion. Cells were spun down at 4C 750 for 5min and resuspended in 3ml of 1X RBC lysis buffer (eBioscience 00-4333, or diluted from eBioscience 00-4300) followed by 4-5 min incubation at RT. 10ml of 1X PBS was then added. The cells were then spun down at 4C at 750 for 5min, resuspended in 5ml of 2% FBS in 1X PBS, filtered through a 40um strainer, spun down again iat 4C 750g for 5min, resuspended in 50-200ul of 2% FBS in PBS, and spun down again. Cells were loaded according to the standard protocol of the Chromium single cell 5' V1 kit