Instrument: Illumina HiSeq 2500
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: PAIRED
Construction protocol: Cells of each embryo were dissociated with 300 ul 1% trypsin in Ca2+-Free ASW with 5 mM EGTA for 5 min. Embryos were pipetted 5 min on ice to complete dissociation of individual cells. 500 ul ice cold Ca2+-Free ASW + 0.5% BSA was added to terminate digestion. Cells were collected by centrifuging at 900g for 5 min at 4 C and then resuspended in 50 ul ice cold Ca2+-Free ASW + 0.5% BSA. Single cell suspensions were loaded onto the 10x Genomics Chromium system using Chromium Single Cell 3 Library and Gel Bead Kit v2 (Lot. 120267) to generate and amplify cDNAs, as recommended by the manufacturer (10X Genomics, CA). Illumina sequencing libraries were generated from the cDNA samples using the Nextera DNA library prep kit (Lot. FC-121-1030) and sequenced using Illumina HiSeq 2500 Rapid flowcells (Illumina Inc., CA) with paired-end 26nt + 125nt reads following standard Illumina protocols.