Placental infection single-cell and single-nuclei maps
Placental infection single-cell and single-nuclei maps
All tissue samples were obtained with written informed consent from all participants in accordance with the guidelines in the Declaration of Helsinki 2000. Placental samples were obtained from elective terminations from The MRC and Wellcome-funded Human Developmental Biology Resource (HDBR, http:// www.hdbr.org), with appropriate maternal written consent and approval from the Fulham Research Ethics Committee (REC reference 18/LO/0822). The HDBR is regulated by the UK Human Tissue Authority (HTA: www.hta.gov.uk) and operates in accordance with the relevant HTA Codes of Practice.  Explants were treated with Corning® Cell recovery solution (Corning, CLS354253) for 15 minutes on ice to remove Matrigel® remnants. The plate was shaken every 5 minutes to dislodge the explant from the membrane. The tissue was transferred to a 50mL Facon tube with ice-cold PBS and washed by inverting, then transferred to a Petri dish with fresh ice-cold PBS, and the villi were gently scraped using forceps and scalpel. Using a Pasteur pipette with a cut tip, all of the tissue was transferred to a Falcon tube with 40 mL of pre-warmed 0.2% trypsin-EDTA and sealed with parafilm. Digestion was performed at 37˚C on a rotator with gentle rotation at 30 rpm for 7 minutes and stopped with 2mL of PBS. The tissue was filtered using a 100µm strainer and the strainer was washed with 5-10 mL of RT PBS (the tissue on the filter was kept for the second step of digestion). The filtrate was centrifuged at 200g for 5 minutes at RT. The supernatant was discarded and the pellet kept on ice (digest 1). The undigested tissue was retrieved from the filter by forceps and placed in a 50 mL Falcon tube containing 12.5mL of prewarmed 1mg/ml Collagenase V (Sigma-Aldrich, C9263) and 40µg/mL DNase I (Roche, 10104159001) in PBS. The tube was sealed with Parafilm and transferred to a tube rotator in the incubator. The tissue was digested for 30 minutes with slow rotation at 30 rpm. The cell suspension was filtered through a 100µm filter and the filter was washed with 5mL of RT PBS. The filter with any undigested tissue was discarded. Cells were pelleted at 200g for 5 minutes at RT (digest 2). Cells from digest 1 and digest 2 were pooled and washed with PBS with 20µg/mL of DNase I (DNase I buffer). Cell suspension was centrifuged at 200g for 5 minutes at RT, the pellet resuspended in 1mL of DNase I buffer and kept on ice. Cells were counted using a haemocytometer.   To remove erythrocytes and debris, a Pancoll gradient was used. The cell suspension was topped up to 10mL with DNase I and very slowly added on top of 5mL of Human Pancoll (PAN-Biotech, P04-60500) in a 15mL Falcon tube. The suspension was centrifuged at 600g for 20 minutes at RT, with using acceleration=0, break=1 settings on the centrifuge. Cells were collected from the interface using a Pasteur pipette. The cells were washed with DNase I buffer and centrifuged at 200g for 5 minutes at RT twice. Cells were passed through a 35-40µm filter and counted (bulk cells). 1 million of these bulk cells were collected for flow cytometry assessment (see Flow cytometry section).  To enrich the final cell suspension for the CD45+ population, 1 million of bulk cells were separated and kept on ice and the rest was submitted to the MACS-based enrichment following the CD45 MicroBeads protocol (product code). The cells were centrifuged at 200g for 5 minutes at RT and the pellet resuspended in DNase I buffer and counted (CD45 cells). Bulk cells and CD45 cells were mixed in 1:1 ratio in PBS/0.04% BSA prior to loading of the 10x Genomics Chromium inlet.   Three independent placental tissue regions per donor were excised and processed to single cell suspension. For hashtag antibody staining, 500,000 cells from bulk and CD45+ enriched fraction were individually re-suspended in Cell Staining Buffer (420201, Biolegend) and blocked with 5 µL Human TruStain FcX™ (422301, Biolegend) for 10 minutes at 4 °C. Each of the cell suspensions originating from different placental tissue regions were hashtagged with different TotalSeq-B™ antibodies for 30 minutes at 4 °C. 1 µg of the following TotalSeq-B™ antibodies were used; TotalSeq™-B0253 anti-human Hashtag 3 Antibody (394635, Biolegend), TotalSeq™-B0254 anti-human Hashtag 4 Antibody (394637, Biolegend) and TotalSeq™-B0255 anti-human Hashtag 5 Antibody (394639, Biolegend). Antibody-tagged cells were washed three times with 3 ml of Cell Staining Buffer using centrifugation speed of 500 x g for 5 minutes at 4°C. Bulk cells and CD45 cells were mixed in 1:1 ratio then filtered through a 40 uM cell strainer. Cells were counted and diluted to desired loading concentration to recover between ~4000 cells. Hashtagged cells were pooled at equal cell number ratio (targeted for a total of ~12000 cells) right before loading of the 10x Genomics Chromium inlet.  Cells and nuclei were loaded into the 10x Genomics Chromium inlet according to the manufacturer's instructions. The Chromium NEXT GEM Single Cell 3′ Library v3.1 chemistry Dual index (10x Genomics) was used and libraries were prepared according to manufacturer's instructions aiming to recover between 2,000 and 25,000 cells per reaction. For cell hashing workflow, Chromium NEXT GEM Single Cell 3′ Library v3.1 with Feature Barcoding technology for Cell Surface Protein was used according to the manufacturer's protocol (10x Genomics).