GSM4227076: SSEA4+P-S3; Sus scrofa; RNA-Seq
Fractions with high purity of SSEA4-positive spermatogonia were resuspended with Trizol (Invitrogen, USA) and total RNA was extracted for further analysis. RNA seq:total RNA was used for removing rRNAs with NEBNext rRNA Depletion Kit (New England Biolabs, Inc., Massachusetts, USA) following the manufacturer's instructions. RNA libraries were constructed by using NEBNext® Ultra™ II Directional RNA Library Prep Kit (New England Biolabs, Inc., Massachusetts, USA) according to the manufacturer's instructions. Libraries were controlled for quality and quantified using the BioAnalyzer 2100 system (Agilent Technologies, Inc., USA). Library sequencing was performed on an illumine Hiseq instrument with 150bp paired end reads. Paired-end reads were harvested from Illumina Hiseq-4000 sequencer, and were quality controlled by Q30. After 3' adaptor-trimming and low quality reads removing by cutadapt software(v1.9.3), the high quality clean reads were aligned to the reference genome (susScr3) with hisat2 software (v2.0.4). Then, guided by the Ensembl gtf gene annotation file, cuffdiff software (part of cufflinks) was used to get the gene level FPKM as the expression profies of mRNA, and fold change and p-value were calculated based on FPKM, differentially expressed mRNA were identified.
GEO Accession
GSM4227076