GSM6422720: P0, Nkx2.1-CTX, CREST_bulk, biol rep 3, library 4; Mus musculus; OTHER
Tissues, organoids and cultured cells were dissociated using papain (Worthington), Accutase (Gibco) or Trypsin-EDTA accordingly, and then pipetted into single-cell suspensions in collection medium [Neurobasal Medium, supplemented with 5% FBS]. Cells were filtered (Falcon 5mL Round Bottom Polyst) and then sorted by FACS to collect EGFP+/BFP+ cells for genomic DNA extraction (Vazyme) for bulk amplification or for single-cell RNA sequencing (10x Chromium System V3 or V3.1). Single-cell RNA sequencing library were performed according to the manufacter's instructions (single cell 3' v3 protocol, 10x Genomics). CREST barcode libraries were amplified from the single-cell cDNA libraries generated in step 2.3 of the 10X Chromium single cell 3' protocol with two rounds of PCR. Briefly, CREST_sc_1st_F and CREST_sc_1st_R primers were first used to enrich CREST barcodes tagged with 10X cell barcodes and UMIs. PCR products were purified using a 2-step bead purification process (0.5x then 1.1x) and then used as templates for a second round of PCR to attach sequencing adaptors and indices with CREST_sc_2nd_F and CREST_sc_2nd_R1/2 primers. The second-round PCR products (single-cell CREST libraries) were purified using a gel purification kit (Vazyme) and DNA of the targeted size collected.  For bulk amplification of CREST barcodes, both V1 and V2 CREST barcodes were enriched together from genomic DNA using three rounds of PCR amplification. Specifically, UMI-indexing PCR was first performed to identify CREST DNA from individual cells using the CREST_bulk_1st_F primer (containing 39 bp of partial Illumina sequencing adaptor, 12 bp of random UMI, and 20 bp of BFP binding sequence [10 bp upstream from CREST barcode]). Up to 500 ng of gDNA was used as template for UMI-indexing PCR for each library (for samples yielding less than 500 ng of gDNA, entire volumes were used as templates). The indexed CREST barcodes were purified using 1.6x VAHTS DNA Clean Beads (Vazyme) and amplified via a second round of PCR (using primers REST_bulk_2nd_F and CREST_bulk_2nd_R). Amplified CREST barcodes were selected and purified using a 2-step bead purification process (0.55x, then 1.45x) according to the manufacturer's recommendations (Vazyme). Finally, sequencing adaptors and sample indices were attached to each library via a third round of PCR (using primers CREST_bulk_3rd_F and CREST_bulk_3rd_R1-8 [containing 8 bp indices]). PCR products were purified using 1.6x VAHTS DNA Clean Beads.