GSM6831757: Tracer TLS #1 - scRNAseq; Mus musculus; RNA-Seq
Single-cell RNAseq (scRNA-seq) experiments have been performed as previously described (Veenvliet, Bolondi et al 2020). Briefly, Tracer TLS were generated as described above. Two individual 120h TLS (Tracer TLS 1 and 2) were selected based on the presence of one axis of elongation, clear formation of a neural tube domain and segmentation. Individual structures were picked with a p200 with the pipette tip cut-off at the 50µl mark, and serially washed through pipette transferring (cut 200µl tip) in wells filled with 200µl of 1xPBS/0.4%BSA (5 transfers) to get rid of the Matrigel. Each structure was then dissociated in 200µl TrypLE Express (Gibco) for 25 minutes at 37ºC, with pipetting every 5 minutes intervals. The cell suspension was filtered using Scienceware Flowmi Cell Strainers, 40µm. Cells were washed twice with 1ml 1xPBS/0.4%BSA with centrifugation steps performed for 5 minutes at 1200rpm in low DNAbind Eppendorf tubes. The cell concentration was determined using a hemocytometer and cells were subjected to single-cell RNA sequencing (10x Genomics, Chromium™ Single Cell 3' v3.1; one structure per reaction). Single-cell libraries were generated according to the manual, with one modification: fewer PCR cycles (n=8) were ran than recommended during cDNA amplification or library generation/sample indexing to increase library complexity. Quality and concentration of the obtained libraries were measured using Agilent High Sensitivity D5000 ScreenTape on an Agilent 4150 TapeStation. Libraries were sequenced with a minimum of 400 million paired end fragments according to parameters described in the manual. Lineage tracing barcodes (intBCs) libraries were prepared for all samples via PCR amplification from the double stranded 10x cDNA. Briefly, 100ng of 10x cDNA was used to prepare 4x25µl PCR reactions for each sample. Amplification was carried out using Q5 2X MasterMix (NEB; M0492S) with the following steps: 98ºC 30sec/ 98ºC 10sec – 68ºC 30sec – 72ºC 40sec (10 cycles)/ 72ºC 2 minutes/ 4ºC hold. Next, the four reactions for each sample were pooled together and a double-sided AMPure XP beads (Beckman, A63881) cleanup (0.45X/0.7X) was performed to purify the intBCs libraries. Quality and concentration of the obtained libraries were measured using Agilent High Sensitivity D5000 ScreenTape on an Agilent 4150 TapeStation. Libraries were then sequenced using asymmetric end sequencing (150 cycles kit; 28/91 FC-410-1002) on a Novaseq platform at a minimum of 100 million fragments per sample.