title: GSM7075777: E64 Motor-somatosensory, bio rep 1; Macaca mulatta; RNA-Seq
library_construction_protocol: Fetal macaque brains were isolated from E37 to E110, put on a dish containing PBS and sectioned. Telencephalic regions were identified and dissected using a blade. For E37-43 brains, the entire anterior and medio-posterior parts of the hemispheres were cut. Each tissue was incubated with HBSS-Papain (2 mg/ml, BrainBits) from 15 (for the early ages) to 30 (for more advanced ages) minutes at 37oC. The solution was removed and the tissue gently triturated in HBSS-DNase I 0.1mg/ml solution (StemCells) using a 2 ml pipette. Then, samples were filtered through 30 µm strain filters and the cells counted with an automatic cell counter (ThermoFisher Scientific). Samples were diluted in HBSS to 1000 cells/microliter and processed for single cell RNA-seq analysis within 20 min at Yale Center for Genome Analysis (YCGA) core facility. GEM Generation and Barcoding. Single cell suspension in RT Master Mix is loaded on the Single Cell A Chip and partition with a pool of about 750,000 barcoded gel beads to form nanoliter-scale Gel Beads-In-Emulsions (GEMs). Each gel bead has primers containing (i) an Illumina® R1 sequence (read 1 sequencing primer), (ii) a 16 nt 10x Barcode, (iii) a 12 nt Unique Molecular Identifier (UMI), and (iv) a poly-dT primer sequence (30nt). Upon dissolution of the Gel Beads in a GEM, the primers are released and mixed with cell lysate and Master Mix. Incubation of the GEMs then produces barcoded, full-length cDNA from poly-adenylated mRNA.   Post GEM-RT Cleanup, cDNA Amplification and library construction. Silane magnetic beads are used to remove leftover biochemical reagents and primers from the post GEM reaction mixture. Full-length, barcoded cDNA is then amplified by PCR to generate sufficient mass for library construction. Enzymatic Fragmentation and Size Selection are used to optimize the cDNA amplicon size prior to library construction. R1 (read 1 primer sequence) are added to the molecules during GEM incubation. P5, P7, a sample index, and R2 (read 2 primer sequence) are added during library construction via End Repair, A-tailing, Adaptor Ligation, and PCR. The final libraries contain the P5 and P7 primers used in Illumina bridge amplification.