Macs debris removal solution

Uterine–placental interface tissues were harvested from gd 15.5 (n = 3 pregnancies/gd) and 19.5 (n = 4 pregnancies/gd) rat placentation sites and placed in ice-cold Hank’s balanced salt solution (HBSS). Tissues were minced into small pieces with a razor blade and digested with Dispase II (1.25 units/mL, D4693, Sigma-Aldrich), 0.4 mg/mL collagenase IV (C5138, Sigma-Aldrich), and DNase I (80 units/mL, D4513, Sigma-Aldrich) in HBSS for 30 min. Red blood cells were lysed using ammonium-chloride-potassium lysis buffer (A10492-01, Thermo Fisher) with rotation at room temperature for 5 min. Intact cells were washed with HBSS supplemented with 2% fetal bovine serum (Thermo Fisher), and DNase1 (Sigma-Aldrich) and passed through a 100-µm cell strainer (100ICS, Midwest Scientific). Following enzymatic digestion, cellular debris was removed using MACS Debris Removal Solution (130-109-398, Miltenyi Biotec). Cells were then filtered through a 40-µm cell strainer (40ICS, Midwest Scientific) and cell viability was assessed, which ranged from 90 to 93%. Cells were used for the preparation of single cell libraries using the 10× Genomics Chromium system (10× Genomics) and sequenced with an Illumina NovaSEq. 6000 (Illumina) by the KUMC Genome Sequencing Facility.

Uterine-placental interface tissue (also called metrial glands) were dissected from gd 15.5 (n=3 pregnancies) and 19.5 rat placentation sites (n=3 pregnancies) as previously described [20 (link),76 (link)] and put in ice cold Hank’s balanced salt solution (HBSS). Tissues were minced into fine pieces with a razor blade and digested in Dispase II (1.25 units/mL, D4693, Sigma-Aldrich), 0.4 mg/mL collagenase IV (C5138, Sigma-Aldrich), and DNase I (80 units/mL, D4513, Sigma-Aldrich) in HBSS for 30 min. Red blood cells were lysed using ACK lysis buffer (A10492–01, Thermo-Fisher), rotating at room temperature for 5 min. Samples were washed with HBSS supplemented with 2% fetal bovine serum (FBS, Thermo-Fisher), and DNase1 (Sigma-Aldrich) and passed through a 100 μm cell strainer (100ICS, Midwest Scientific). Following enzymatic digestion, cell debris was removed using MACS Debris Removal Solution (130–109-398, Miltenyi Biotec). Cells were then filtered through a 40 μm cell strainer (40ICS, Midwest Scientific) and cell viability was assessed, which ranged from 90 to 93%.