GSM8068146: FT4; Homo sapiens; RNA-Seq
Endometrial and decidual tissues were grossly dissected and washed in PBS, then finely minced before being digested in a shaking 37°C water bath for 15-30 mins in sterile medium containing 0.28WU/mL Liberase TM (Roche, Catalog  # 05401127001) and 30ug/mL DNase (Roche, catalog # 10104159001)]. A large-bore transfer pipet was used to pipet tissue up and down during digestion to break down tissue. Digested tissue was pelleted, subjected to RBC lysis, and then filtered through a 70μm filter. For the fresh/unfrozen tissue, cells were immediately processed within 2 hours for flow sorting. Frozen samples were similarly processed within 2 hours, and frozen in cryotubes at a concentration of 1x107/mL in 1mL of freezing medium, consisting of 90% RPMI (ThermoFisher, Catalog # 11875085), 10% DMSO (Sigma, Catalog # D2650-100mL), and 1mg/mL BSA (Sigma, A9418)]. Samples were frozen in a Mr.Frosty freezing container for 24 hours before being placed in a -120℃ freezer for up to 6 months. Immediately prior to flow sorting, cryovials were thawed for 30 seconds in a 37ºC water bath and immediately washed in PBS to remove DMSO. Next-generation sequencing libraries were prepared using the 10× Genomics Chromium Single Cell 3' Library and Gel Bead kit v3, per manufacturer's “Chromium Single Cell Reagent Kits” protocol. Libraries were uniquely indexed using the Chromium i7 Sample Index Kit, pooled, and sequenced on the Illumina NovaSeq 6000 sequencer (v1.5 chemistry) in a paired-end, single indexing run. Sequencing targeted 20,000 mean reads per cell.