normalization data transformation protocol
nucleic acid sequencing protocol
Libraries were sequenced aiming at an average depth of 1 million reads/cell, on an Illumina HiSeq 2000 with v4 chemistry (paired-end 75-bp reads).
sample collection protocol
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 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 single-cell RNA-seq using the plate-based SS2 protocol, we created overlapping gates that comprehensively and evenly sampled all immune cell population in the decidua. B cells (CD19+ /or CD20+) cells were excluded from our analysis, due to its absence in decidua. Single cells were sorted into 96-well full-skirted Eppendorf plated chilled to 4C, prepared with lysis buffer consisting of 10 ul of TCL buffer (Qiagen) supplemented with 1% b-mercaptoethanol. Single-cell lysates were sealed, vortexed, spin down at 300g at 4C for 1 min, immediately placed on dry ice, and transferred for storage at -80C. The SS2 protocol was performed on single-cells as described previously, with some modifications. Libraries were sequenced aiming at an average depth of 1 million reads/cell, on an Illumina HiSeq 2000 with v4 chemistry (paired-end 75-bp reads).
nucleic acid library construction protocol
Libraries were made using Nextera XT
nucleic acid extraction protocol
RNA was extracted, cDNA created and amplified, as part of the smart-seq 2 protocol.