title: Illumina NovaSeq 6000 paired end sequencing; Single cell RNA sequencing (scRNA-seq) of human embryoid body (day 4 of culture)
design: Single cell RNA sequencing (scRNA-seq) of human embryoid body (day 4 of culture)
library_construction_protocol: Naive human induced pluripotent stem cell (hiPSC) (PB12.CO3: 46XX) were plated in ultra-low attachment U-bottom 96 well plates at a density of 8 000 to 10 000 cells/well in a PGC-LC medium. This latter was composed of Glasgow's Minimal Essential Medium (GMEM, GIBCO), 15% KSR (GIBCO), 0.1 mM nonessential amino acids (GIBCO), 0.1 mM 2-mercaptoethanol (GIBCO), 100 U/ml Penicillin- 100 U/ml Streptomycin (GIBCO), 2 mM L-Glutamine, 1 mM Sodium pyruvate (Sigma), and the following cytokines: 500 ng/ml BMP4 (Peprotech), 1 µg/ml human LIF (Peprotech), 100 ng/ml SCF (Peprotech), 50 ng/ml EGF (Peprotech), and 10 µM ROCK inhibitor. To force embryoid body aggregation, cells were centrifuged at 200 g for 2 min and cultured for 4 days. Around 60 embryoid bodies by sample were dissociated and stained with Hoescht 33258. Single-cell suspensions from EB at day 4 were pooled before Hoescht 33258 staining. Total viable cells were flow-sorted into PBS-BSA 0.05%. Sorted cells were centrifuged and counted, and 4,000 cells were loaded onto the Chromium 3′ chip. Two replicates (EBD4_A, EBD4_B) from two different pools of EBd4 were encapsulated independently. Cells capture, lysis & RNA extraction were performed using the 10x V3 Chromium technology as recommended by the manufacturer. Around 4,000 cells by replicate were loaded in each lane of the Chromium 3′chip.  Barcoding and library preparation were performed with the 10x Chromium system using version 3 chemistry (Dual index TT_C4 for EBD4_A and TT_C4 for EBD4-B) ), following the manufacturer's protocol. cDNA was synthesized and amplified as per manufacturer's protocol and amplified to construct an Illumina sequencing library. cDNA and libraries were checked for quality on using Agilent with DNA HS chip..