title: GSM7755902: Sacral-derived cells, post-umbilical gut replicate 2; Gallus gallus; RNA-Seq
library_construction_protocol: At embryonic day 10, the gastrointestinal tract, including the associated Nerve of Remak and pelvic plexuses , was dissected from chick embryos and washed with Ringer's solution. Pre- and post-umbilical regions were separated, broke into pieces in chilled DPBS and loose-fit homogenized in Accumax solution (EMD Millipore). 400ml of Accumax-tissue mixture was aliquoted into 1.7 ml Eppendorf tubes and shaken at 37 °C for 12mins. After dissociation, chilled Hanks Buffered Saline Solution (HBSS) supplemented by BSA (125mg in 50ml, Sigma; 0.2% w/v) and 1M HEPES (500ml in 50ml, PH7.5, ThermoFisher) was added to quench the reaction. The dissociated cells were passed through a 70mm cell strainer (Corning) and collected by centrifuging at 500g for 11mins at 4 °C. The cells were resuspended in HBSS-BSA, supplemented 7-AAD Viability Staining Solution (Biolegend # 420404, 500 TESTS), and sorted for YFP+, viable single cells using Sony SY3200 cell sorter at the Caltech Flow Cytometry Facility. Library contrusted per manufacturers instructions. The library was sequenced on NovaSeq S4 lane with 2x150bp reads by Fulgent Therapeutic