title: GSM8411285: mouse_mirmOTSTEO_sample_18_a (control); Mus musculus; RNA-Seq
library_construction_protocol: Upon reaching the desired developmental stage (8 cell (E2.5) or 32 cell (E3.5)), several control and mimic-treated mouse embryos (3-5 embryos/group/timepoint) were collected for single cell dissociation and isolation. Embryos were briefly transferred into Tyrodes solution (Millipore, ref # MR-004-D) to digest the zona pellucida, then were dissociated through trituration in TrypLE (Life Technologies, ref # 12604-013) and picked with fine glass capillaries. Individual cells were dispensed in 4.1 uL Smart-Seq2 lysis buffer and stored at -80°C until library preparation. Following removal from -80°C storage and thawing, cell lysis (thermocycler, 72°C for 3 minutes) and reverse transcription were performed. This was followed by two polymerase chain reactions (PCRs), the first to amplify the cDNA product (20 cycles) and second with indexes to barcode the libraries for multiplexing (11 cycles). Sample purification, downstream library preparation, sequencing with a NextSeq550 and data analysis using in-house pipelines were all performed as described in Petropoulos et al., 2016