Tracking Early Mammalian Organogenesis – Prediction and Validation of Differentiation Trajectories at Whole Organism Scale (Complementary data)
Tracking Early Mammalian Organogenesis – Prediction and Validation of Differentiation Trajectories at Whole Organism Scale (Complementary data)
Mouse embryos were collected under the UK Home Office project licence number PP9552402. Animals used in this study were 6-16 week-old wild type females of a mixed (CBAxC57BL/6)F2 background, maintained on a lighting regime of 14h light and 10h darkness with food and water supplied ad libitum. Following timed mating crosses between transgenic mTmG (B6.129(Cg)Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo /J [Muzumdar et al., 2007]) males and wildtype females, females were killed 7 days after a vaginal plug was observed via a Schedule 1 method. The uterine tissue and the decidua were cut away to extract the E7 embryo. The Reichert's membrane was peeled off and embryos were staged according to [Downs and Daviers, 1993]. Early bud (EB) stage embryos were taken for further analysis. For primitive streak dissections, the extra-embryonic part of the conceptus was cut off, the anterior and posterior embryonic portions were separated, and the posterior portion flattened out, taking care not to mix up the orientation. The posterior portion containing the primitive streak was cut at the halfway point and each half was halved again creating 4 similar-sized primitive streak segments. All embryo and primitive streak dissections were performed in Dulbecco's Phosphate Buffered Saline (PBS; with CaCl2 and MgCl2, Gibco) supplemented with 10% FCS (Gibco), 50U/ml penicillin and 50U/ml streptomycin. For scRNA-seq of primitive streak cells, individual primitive streak regions were collected in separate Eppendorf tubes in FACS buffer (PBS without CaCl2 and MgCl2 (Gibco) supplemented with 10% FCS (Gibco), 50U/ml penicillin and 50U/ml streptomycin).  After centrifugation at 200G for 5 min, tissues were resuspended in 200-250ul of TrypLE Express (Life Technologies) and incubated at 37C for 7 min with regular agitation and dissociated by pipetting. Cells were washed with 1ml of FACS buffer, centrifuged at 200G for 5min and resuspended in FACS buffer with Hoechst 33358 for sorting. In order to reduce cell loss from these small cell populations, wild type adult mouse thymocytes were added to the samples and were gated out based on their typical FSC-SSC profiles and absence of tdTomato expression. Each primitive streak region yielded on average 200 cells. Cells were kept on ice throughout the procedure. Individual cells were sorted directly into 96 well plates (Starlab E14031200) into 2.3ul of lysis buffer containing SUPERase-In RNase Inhibitor 20U/ul (Ambion, AM2694 ), 10% Triton X-100 (Sigma, 93443 ) and RNAse-free water. Plates were processed using a combination of Smart-Seq2 [Picelli et al., 2014] and mcSCRB-seq [Bagnoli et al., 2018] For detailed protocol see [Sturgess et al., 2021]. Single cells were lysed in a buffer that contains free dNTPs and oligo(dT)-tailed oligonucleotides with a universal 5'-anchor sequence. Following nucleic acid extraction, RT is performed, which adds 2–5 untemplated nucleotides to the cDNA 3′ end. A template-switching oligo (TSO) is added, carrying 2 riboguanosines and a modified guanosine to produce a locked nucleic acid (LNA) as the last base at the 3′ end. After the first-strand reaction, the cDNA is amplified using a limited number of cycles. Next, tagmentation is used to construct sequencing libraries quickly and efficiently from the amplified cDNA.