GSM8110667: E14 chick embryonic hindgut; Gallus gallus; RNA-Seq
Single cell suspensions were generated by first dissecting guts in ice-cold 1X PBS. Whole guts were placed in DMEM + 10% chick embryo extract at 37°C until all samples were dissected and ready for dissociation. For pPIDNB experiment samples, dissected guts were observed under epifluorescence to isolate regions with mScarlet+ transgenic cells. 2-3mm gut segments, 1 from each replicate, were then incubated at 37°C in 1mL dispase for 10min as for bulk RNA sequencing sample preparation, followed by 1 PBS wash. Gut pieces (pooled by condition) were placed in the open caps of Eppendorf tubes containing 500µL 0.25% Trypsin-EDTA (Sigma) and mechanically disrupted using forceps. Tubes were then closed and inverted before placing samples at 37°C for 12-15min. Samples were mechanically disrupted with P1000 and P200 pipettes every 2min during the first half of the incubation, and with a 1mL syringe/25G needle during the second half.  When pPIDNB midgut, pPIDNB-HOXD13 midgut, and electroporated control hindgut suspensions were homogenous with no visible tissue clumps, 400µL of ice cold DMEM + 4% FBS was added to each sample, which was then strained using a 5mL polystyrene round-bottom tube with a 35µm nylon mesh cell strainer cap. After one spin at 800xg for 5min at 4°C, samples were resuspended in DMEM + 4% FBS, labeled for 5min with DAPI, and placed on ice. To enrich for mScarlet+ cells, midgut samples were iteratively sorted 3 times for approximately 30min each using an On-chip Sort microfluidic chip sorter. The mScarlet+mNeon+DAPI- population was gated to select for living cells with induced transgene expression. Trypan blue staining before and after all samples were sorted confirmed minimal cell death due to time spent on ice. After straining again through a 70µm mesh strainer and cell counting using a hemocytometer, sorted suspensions in 1X PBS were used for GEM generation according to the 10X Chromium Next GEM Single Cell v3.1 (Dual Index) protocol 10X scRNA-seq v3.1 chemistry (Dual-index)