title: GSM5816044: YN, Embryo 3, ICM; Equus caballus; RNA-Seq
library_construction_protocol: Each hemi-embryo was transferred to a DNA LoBind tube (Eppendorf) with extraction buffer from PicoPure RNA isolation kit (Applied Biosystems) and incubated for 30min at 42°C prior to storage at -80°C. The extraction of hemi-embryos RNA was performed using the PicoPure RNA isolation kit (Applied Biosystems), including a DNAse treatment, following the manufacturer's instructions. RNA quality and quantity were obtained with 2100 bioanalyzer system using RNA 6000 Pico or nano kit (Agilent Technologies) according to the manufacturer's instructions. The RNA Integrity Number median was 9.6 (range from 9.0 to 9.9). Five nanograms of total RNA (except YN_E1 for which only 2.5ng were used) were mixed with ERCC spike-in mix (Thermofisher) according to the manufacturer recommendations and then used for re-transcription and amplification using the SMART-Seq V4 ultra low input RNA kit (Clontech) according to the manufacturer recommendations. Nine PCR cycles were performed for each hemi-embryo. cDNA quality was assessed on an Agilent Bioanalyzer 2100, using an Agilent High Sensitivity DNA Kit (Agilent Technologies). Libraries were prepared from 0.15 ng cDNA using the Nextera XT Illumina library preparation kit (Illumina). Libraries were pooled in equimolar proportions and sequenced (Paired-end 50-34 pb) on NextSeq500 instrument, using a NextSeq 500 High Output 75 cycles kit (Illumina).