title: Sample 9
organism: Homo sapiens
description: Protocols: Placental and decidual tissue for immunohistochemistry were obtained from elective terminations of normal pregnancies at Addenbrooke's Hospital between 6 and 12 weeks gestation. Decidual and placental tissue were washed in HAMS F12 medium, macroscopically separated and then washed for at least 10 mins in RPMI or HAMS F12 medium respectively before processing.Decidual tissues were chopped using scalpels into approximately 0.2 mm3 cubes and enzymatically digested in 15ml 0.4mg/mL collagenase V (Sigma, C-9263) solution in RPMI 1640 medium (ThermoFisher Scientific, 21875-034)/10% FCS (Biosfera, FB-1001) at 37°C for 45 min. The supernatant was diluted with medium and passed through 100um cell sieve (Corning, 431752) and then 40um cell sieve (Corning, 431750). The flow-through was centrifuged and resuspended in 5ml of red blood cell lysis buffer (Invitrogen, 00-4300) for 10min.Each first trimester placenta was placed in a petri dish and the placental villi were scraped from the chorionic membrane using a scalpel. The stripped membrane was discarded and the resultant villous tissue was enzymatically digested in 70 ml 0.2% trypsin 250 (Pan Biotech P10-025100P)/0.02% EDTA (Sigma E9884) in PBS with stirring at 37°c for 9 min. The disaggregated cell suspension was passed through sterile muslin gauze (Winware food grade) and washed through with Hams F12 medium (Biosera SM-H0096) containing 20% FBS (Biosera FB-1001). Cells were pelleted from the filtrate by centrifugation and re-suspended in Hams F12. The undigested, gelatinous tissue remnant was retrieved from the gauze and further digested with 10-15 ml collagenase V at 1.0mg/ml (Sigma C9263) in Hams F12 medium/10% FBS with gentle shaking at 37°C for 10 min. The disaggregated cell suspension from collagenase digestion was passed through sterile muslin gauze and the cells pelleted from the filtrate as before. Cells obtained from both enzyme digests were pooled together and passed through 100um cell sieve (Corning, 431752) and washed in Hams F12. The flow-through was centrifuged and resuspended in 5ml of red blood cell lysis buffer (Invitrogen, 00-4300) for 10min.Decidual and blood cells were incubated at 4°C with 2.5ul of antibodies in 1% FBS in DPBS without Calcium and Magnesium (ThermoFisher Scientific, 14190136). DAPI was used for live/dead discrimination. We used an antibody panel (Fig xxx) designed to enrich for certain population for single-cell sorting and single-cell RNA sequencing (scRNA-seq). Cells were sorted using a Becton Dickinson (BD) FACS Aria Fusion with 5 excitation lasers (355nm, 405nm, 488nm, 561nm and 635nm Red), and 18 fluorescent detectors plus forward and side scatter. The sorter was controlled using BD FACS DIVA software (version 7).For the droplet RNA-seq methods, blood and decidual cells were sorted into immune (CD45+) and non-immune (CD45-) fractions. Only viable cells were considered, and B cells (CD19+/CD20+) cells were excluded from our analysis. Placental cells were stained for DAPI and only viable cells were sorted. Cells were sorted into an eppendorf containing PBS with 0.04% BSA. Cells were immediately counted using a Neubauer hemocytometer and loaded in the 10x-genomics machine. Tissue DNA and RNA were extracted from fresh frozen samples using the AllPrep DNA/RNA/miRNA kit (Qiagen) following the manufacturer's instructions. Libraries for whole-genome sequencing were constructed according to Illumina library protocols
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2018-10-18T17:04:01Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2018-10-04T15:05:09Z
attributes.tag: External Id
attributes.value: SAMEA4952652
attributes.tag: INSDC center name
attributes.value: Wellcome Sanger Institute
attributes.tag: INSDC first public
attributes.value: 2018-10-18T17:04:01Z
attributes.tag: INSDC last update
attributes.value: 2018-10-04T15:05:09Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-7304:Sample 9
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: clinical information
attributes.value: 6 to12 weeks gestation
attributes.tag: common name
attributes.value: human
attributes.tag: developmental stage
attributes.value: adult
attributes.tag: disease
attributes.value: normal
attributes.tag: individual
attributes.value: D9
attributes.tag: organism part
attributes.value: placenta
attributes.tag: sample name
attributes.value: E-MTAB-7304:Sample 9
attributes.tag: scientific_name
attributes.value: Homo sapiens
attributes.tag: sex
attributes.value: female