title: GSM8866095: Uterus Transplant 2_Biopsy 2_L2_2; Homo sapiens; RNA-Seq
library_name: GSM8866095
library_construction_protocol: Endometrial biopsies were collected with a Pipelle® suction curette (Cooper Surgical; Ref. 8200) in the clinical setting and transported to the laboratory in DPBS with calcium and magnesium (DPBS+/+; Gibco™; Cat. 14-040-133) on ice. Multiple passes of the suction curette were performed to acquire sufficient tissue for multimodal analysis. Endometrial biopsies were then digested to isolate cells for downstream analysis including sorting, flow cytometry, or in vitro incubation. Endometrial biopsies used for immunofluorescence microscopy were embedded in O.C.T. mounting media (Tissue-Tek; Cat. No. 4583) in a mold (Tissue-Tek; Cat. No. 4566). Molds were then placed in liquid nitrogen-cooled isopentane (Fisher Scientific; Cat. No. O3551-714 4). Frozen molds were stored at -800C until sectioned onto glass slides for imaging. Endometrial biopsies used for single cell isolation were processed into single cell suspensions for use in downstream single cell isolation techniques, such as scRNA-seq, spectral flow cytometric analysis, and in vitro culture. The biopsies were minced in a petri dish containing 2-3 mL of 370C-warmed DPBS with Calcium and Magnesium (DPBS+/+) on ice with a razor blade or scalpel and transferred to a 50 mL conical for digestion, also on ice. The petri dish was rinsed 4-5 times with 1 mL of 370C-warmed DPBS+/+ which was also transferred to the 50 mL conical. A 1:100 ratio of 5mg/mL Liberase (Sigma Aldrich; 723 SKU 5401127001) and a 1:1000 ratio of 1 mg/mL DNAse I (Sigma-Aldrich; SKU 724 11284932001) was added to the conical and inverted 2-3 times to mix. The conical 725 containing the minced biopsy and digestion media was then placed in a 370C shaking incubator at 240 RPMs and was inspected every 5 minutes to determine if the digestion had completed. If the digestion needed to continue, the conical was inverted 2-3 times to mix and placed back in the 370C shaking incubator at 240 RPMs. Digestion typically took 10-15 minutes on average. The digested sample was placed on ice and filtered through a 70 μm strainer (Stemcell Technologies; Cat. 27260) into a new 50 mL conical. The strainer was rinsed 5 times with 1 mL ice-cold DPBS+/+ before adding more ice-cold DPBS+/+ to the 50mL mark. The digested biopsy was then centrifuged at 300xg for 5 min at 4ºC. After centrifugation, the supernatant was aspirated without disturbing the pellet, which was then transferred to a 15mL conical and resuspended in 2 mL ACK lysis buffer (Quality Biological; Cat. 118-156-101) to incubate on ice for 2 minutes. After ACK lysis, 13 mL ice-cold DPBS without calcium and magnesium (DPBS-/-; Gibco™; Cat. 14-287-739 080) was added to the cell suspension. The 15 mL conical was then inverted 2-3 times to mix and was then centrifuged at 300xg for 5 min at 4ºC. The supernatant was aspirated without disturbing the cell pellet which was then resuspended in 0.5-1mL fresh sort buffer (ice-cold DPBS-/- + 0.04% (w/v) BSA (Jackson ImmunoResearch; Cat. 001-000-162)) for counting. Cells were counted by creating a 1:10 or 1:100 dilution with 0.04% Trypan blue in PBS (Gibco™; Ref. 15250-061). For the 1:10 dilution, 10 μL of the well-mixed cell suspension was added to 90 μL of 0.04% Trypan blue in PBS. For a 1:100 dilution, 10 μL of the 1:10 dilution was added to 90 μL of 0.04% Trypan blue in PBS. Cell counts were performed using a disposable Neubauer improved hemocytometer (INCYTO; DHC-N01-748 5), following the manufacturer's instructions. Live CD45+ cells were used for single-cell RNA sequencing after being FACS-enriched. Cells that were used in CITE-seq experiments were blocked with TruStain Fc block (BioLegend; Cat. 422301) for 30 minutes. Cells were then incubated with TotalSeq B™ Universal cocktail (BioLegend; Cat. 399904) in Cell Staining Buffer (Biolegend; Cat. 420201) and washed following the manufacturer's protocol before being used for scRNA library preparation. Cells isolated for scRNA/CITE-seq were suspended in ice-cold cell sorting buffer. scRNA GEMs and libraries were prepared according to the manufacturer's instructions. 5' libraries were prepared according to 10x Genomics CG000331 protocol, using Chromium Next GEM Single Cell 5' Kit v2 (10x Genomics; PN-1000263). 3' libraries were prepared using 10x Genomics CG000399 protocol, or CG000206 if a CITE-seq antibody panel was used with the appropriate 3' GEM kit (10x Genomics; PN 1000121). Both 3' and 5' libraries used Dual Index Kit TT Set A (10x Genomics; PN 3000431). GEMs were created on either a Chromium Controller or Chromium X (10x Genomics). Either C1000 (Bio-rad) or SimpliAmp (Applied Biosystems) thermocyclers were used for library prep. Quality and concentrations of cDNA and final libraries were calculated from 2100 BioAnalyzer (Agilent) electrograms. Sequencing was performed on a NovaSeq 6000 (Ilumina)
identifiers.namespace: GEO
identifiers.id: GSM8866095_r1