![]() Global analysis of the transcriptome by means of unsupervised clustering indicated that ICM and TE samples clustered to different groups. The correlation of RPKM values for all the genes analysed between any pair of ICM or TE samples was >0.97, indicating a high repeatability of the assay. Among the 22 380 annotated genes, 11 677 and 11 919 we detected as expressed in all ICM and TE samples, respectively. Using the RNASeq algorithms, 77% of reads mapped to annotated transcripts. After discarding duplicated and bad quality reads, an average of 22 508 594 reads per sample was used for analysis. A total of 196 669 501 reads were produced. Sequence analysis was performed using CLC Genomics Workbench and differential expression by DeSeq analysis. Equus caballus genomic sequences and annotation (EquCab2.0) were obtained from the National Center for Biotechnology Information (NCBI). Data analysis was performed using CLC Genomics Workbench. Libraries bearing unique indexes per sample were pooled and sequenced in a single lane of a HiSEqn 2000 apparatus (Illumina) by a single run of 100 bp. Sequencing libraries were prepared using the TruSeq DNA sample preparation kit (Illumina, San Diego, CA, USA). The RNA was then amplified using the SPIA-based Ovation RNASeq-II kit (NuGEN Inc., San Carlos, CA, USA). ![]() Total RNA was extracted from each individual sample using the Arcturus PicoPure RNA isolation kit including DNAse treatment. Individual ICM ( n = 2) and TE ( n = 3) samples were snap frozen in liquid nitrogen and stored at –80☌ until processing. ![]() The ICM were isolated by immunosurgery, whereas polar TE cells were obtained microsurgically. Equine blastocysts were collected by uterine flushing 8 days after ovulation in mares superovulated with reFSH and reLH (Meyers-Brown et al. The objective of this study was to characterise the transcriptome profiles of inner cell mass (ICM) and trophectoderm (TE) cells from horse blastocysts.
0 Comments
Leave a Reply. |