GSM5989964: Gabpb1(hetro_x_hetro)KO-m1; Mus musculus; RNA-Seq
After 96 hours of culture, embryos were transferred to PBS containing 0.1% BSA for washing. Then, zona pellucida was removed in acid Tyrode and the embryos were washed with PBS containing 0.1% BSA. The washed embryo was transferred to a 0.2 ml tube containing 9.5 μl of reaction buffer to make a final volume of 10.5 μl (one embryo in 10.5 μl reaction buffer). After cell lysis in the buffer, 1 μl of the solution was removed and, instead, 1 μl of the diluted ERCC RNA Spike-In Mix was added (Thermo Fisher Scientific, 4456740). The lysed RNA solution with the spike-in RNA were subjected to reverse transcription. The produced cDNA was amplified by PCR with 16 cycles. The amplified cDNA was purified using AMPure XP beads (BECKMAN COULTER, A63882). The purified cDNA was measured on the Bioanalyzer (Agilent) using High Sensitivity DNA Kit (5067-4626) and the normalized volume of DNA was subjected to library preparation using Nextera XT DNA Library Preparation Kit (Illumina, FC-131-1024) by following the vendor's instruction. The obtained library was quality-checked by Bioanalyzer and quantified using Qubit (Thermo Scientific). Paired end sequencing (50 bp + 25 bp) was carried out using the Illumina NextSeq system.