title: GSM5840322: Gastruloid; Mus musculus; RNA-Seq
library_construction_protocol: Gastruloids from NN+CHI, NH+CHI and NH-CHI conditions were generated as described above. From each condition, 28 structures were selected based on the occurrence of a symmetry breaking event and pooled for the single-cell experiment.. Gastruloids were picked with a p200 with the pipette tip cut-off at the 50µl mark, and pooled in a 1,5ml tube filled with ice cold PBS. The three pools were washed twice with ice cold PBS. Next, structures were dissociated in 50µl TrypLE Express (Gibco) for 20 minutes at 37ºC, with pipetting after 10 minutes. A 10x lipid-modified oligonucleotides (LMOs) – Barcode (BC) oligo solution was prepared (1:1; 2µM each) in PBS for each of the three samples on ice. From here on, every step has been performed on ice. After cell dissociation, LMOs – BC solutions were added to each sample separately (final concentration 200nM), and samples were incubated for 5min on ice. In the meantime a 10x co-anchor solution was prepared (2µM) in PBS. Following the 5 min incubation, co-anchor solution was added to each sample (final concentration 200nM), and samples were further incubated for 5min on ice. The reactions was then quenched by adding 1ml PBS+1% BSA to each tube. Cells were then washed twice with 1ml PBS+1% BSA with centrifugation steps performed for 5 minutes at 300g at 4°C in low DNAbind Eppendorf tubes. Next, cell pellets were resuspended in 100ul PBS+0,4% BSA and pooled together in one new tube. The cell suspension was filtered using Scienceware Flowmi Cell Strainers, 40µm. Cells where then centrifuged for 5 minutes at 300g at 4°C, and resuspended in 45ul PBS+0,4% BSA and The cell concentration was determined using a hemocytometer. Cells were then subjected to single-cell RNA sequencing (10x Genomics, Chromium™ Single Cell 3' v3; one reaction). Single-cell libraries were generated according to the manual, with one modification: MultiSeq additive primer (5'-CTTGGCACCCGAGAATTCC-3') was added at the cDNA amplification step (see McGinnis, Patterson et al 2019 for detailed protocol). During amplified cDNA cleanup, the MultiSeq BCs fraction was isolated and a separate library was performed as described in McGinnis, Patterson et al 2019. cDNA library was sequenced with a minimum of 400 million fragments according to parameters described in the manual and MultiSeq BCs library was sequenced with a minimum of 50 million fragments. Single-cell transcriptome profiling using MultiSeq