GSM8244094: ENDO_sample102; Homo sapiens; RNA-Seq
An endometrial biopsy was obtained from each study participant using Pipelle-de-Cornier (Laboratoire CCD, 1103000) just before their laparoscopy. After removal from the uterus, endometrial biopsies were immediately processed into single-cell suspensions and cryopreserved. Briefly, each endometrial biopsy was washed twice in PBS, cut into small pieces with a scalpel and digested in 5 ml of IMDM (Gibco, 31980022) supplemented with 1 mg ml-1 Collagenase type I-A (Sigma-Aldrich, C2674) and 1 mg ml-1 Collagenase type II (Worthington, LS004174) at 37°C for 30-60 minutes. During digestion, the tissue containing tube was inverted several times every 10 minutes until tissue pieces appeared visually dissociated. Enzymatic dissociation was terminated by addition of 5 ml 4°C PBS. Cells were mechanically dissociated in a gentleMACS™ C Tube (Miltenyi Biotec, 130-093-237) using the gentleMACS™ Dissociator (Miltenyi Biotec, 130-093-235), with the Program mLung 02.01. The resulting cell suspension was filtered through a 100µm and 40µm strainer (Corning, 352340 and 352360) and rinsed with an additional 30 ml 4°C PBS. To remove red blood cells, cell suspension was pelleted by centrifugation (300g, 10 min), suspended in 6 ml of 1x red cell lysis buffer (Invitrogen, 00-4300-54) and incubated for 5 min at room temperature. The lysis was stopped by the addition of 35 ml 4°C PBS and endometrial cells were filtered using a 20µm strainer (pluriSelect, 43-50020-01). Following centrifugation (300g, 10 min), the cells were cryopreserved in 90% FCS (Sigma-Aldrich, F7524), 10% DMSO  (Sigma-Aldrich, D8418), frozen in a Bicell freezing vessel (Nihon Freezer, 1-6263-01) at -80°C and subsequently stored long term in liquid nitrogen. Selected endometrial single-cell aliquots for 10x capture were thawed in a 37°C water bath and suspended in 37°C IMDM supplemented with 10% FCS. After centrifugation (300g, 5 min), cell suspension was washed with 4°C PBS + 0.2% BSA (Sigma, A4503). After centrifugation (300g, 5 min), the cell pellet was carefully resuspended in 4°C PBS + 0.2% BSA) to a concentration of  ~1*10^6 cells ml-1 and  strained using a 35µm nylon mesh (Corning, 352235) or a 40µm Flowmi cell strainer (Sigma-Aldrich, BAH136800040) to ensure the absence of cell aggregates. Cell concentration and viability (propidium iodide-negative population) was measured after 5 min incubation of cell aliquot in 2 µg ml-1 propidium iodide (Sigma, 81845) using the portable flow cytometer Moxi Flow (ORFLO Technologies, cassette Typ MF-M, MXC021). Only samples with a post-thawing viability of >70% were included for single-cell capture.  Single-cell capture and cDNA library generation were performed with the 10x Genomics Chromium Single Cell Gene Expression workflow (Chromium NextGEM Single Cell 5' Library and Gel Bead Kit v1.1, Chromium Controller). 25000 viable cells per sample were captured in a single reaction. cDNA libraries were balanced by shallow iSeq and sequenced with a NovaSeq 6000 System (Illumina, S4, 2 x 100 cycles).