Comment[GEAAccession] E-GEAD-418 MAGE-TAB Version 1.1 Investigation Title Transcriptional profiling of pre-granulosa cells Experiment Description To unveil gene expression changes in pre-granulosa cells during their differentiation, we performed RNA sequencing using pre-granulosa cells isolated from embryonic day 17.5, postnatal day1, and 3 mice ovaries. Experimental Design development or differentiation design Experimental Factor Name cell type Experimental Factor Type cell type Person Last Name Muraoka Saga Person First Name Masafumi Yumiko Person Affiliation Mammalian Development Laboratory Department of Gene Function and Phenomics, National Institute of Genetics Person Roles submitter submitter Public Release Date 2021-09-10 Protocol Name P-GEAD-761 P-GEAD-762 P-GEAD-763 P-GEAD-764 P-GEAD-765 Protocol Type sample collection protocol nucleic acid extraction protocol nucleic acid library construction protocol nucleic acid sequencing protocol normalization data transformation protocol Protocol Description Ovaries were treated with MitoTracker Orange probe diluted in culture medium to a final concentration of 1 uM for 5 min and washed with PBS for 5 min three times. Then, GFP single (pre-granulosa cells) or GFP/MitoTracker double positive cells (ovarian epithelial cells as a control) were isolated by FACS. Total RNA was extracted from granulosa cells and epitherial cells in the mouse ovary isolated by florescence activated cell sorting using the RNeasy Micro kit (Qiagen). Then, amplification of total RNA was performed by the Target Amp 1-Round aRNA Amplification Kit103 (epicentre). These amplified samples were used to make a RNA-seq library using the KAPA Stranded mRNA-Seq Kit Illumina platform (KAPA) and barcoded with DNA adapters using the KAPA single-indexed adaptor kit (KAPA). The AMpure XP (Beckman coulter) was used to clean up the synthesized library. Quality assessment was performed using the Agilent DNA1000 Kit (Agilent). The libraries of each sample were sequenced as 100-bp pair-end on an Illumina Hiseq2500 (Illumina) according to the manufactures protocol. Quality control (QC) of raw paired-end (PE) reads for all libraries was performed in the following 3 steps to obtain clean reads: The raw reads and processed reads in each step were checked using FastQC (version 0.11.7, http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Step1: low-quality reads were removed, poly-A/T tails were trimmed from the 3'-end of reads, and reads shorter than 36 bases were removed using PRINSEQ-lite (version 0.20.4). Step2: a fasta file containing adapter sequences was downloaded from the trimmomatic/adapters page of github (https://github.com/timflutre/trimmomatic/tree/master/adapters). Low-quality sequences, adapters, and other Illumina-specific sequences from the reads were trimmed and removed using trimmomatic (version 0.38) \"Paired end mode\". Step3: The same process as in step 1 was repeated. To map reads to the mouse reference genome, the mouse reference genome and gene annotation GTF (UCSC mm9 downloaded from Illumina iGenomes;https://support.illumina.com/sequencing/sequencing_software/igenome.html) were used. Splicing-site and exon information were extracted from the gene annotation GTF file using the Python scripts hisat2_extract_splice_sites.py and hisat2_extract_exons.py, respectively, from the HISAT2 (version 2.1.0) package. The index of the reference genome and annotated transcripts was built using hisat2-build. Clean reads were then mapped to the indexed reference genome using HISAT2 (version 2.1.0). Only uniquely mapped PE reads were extracted from the resulting output and converted into the BAM format file using SAMtools (version 1.7). The obtained BAM files were sorted by genomic coordinates using the SAMtools \"sort\" command. Raw read counts by gene were calculated using the htseq-count from the HTSeq framework (version 0.10.0). The calculation of normalized counts, the detection of differentially expressed genes and statistical analysis were performed using DESeq2 with the cut-off criteria of log2 (fold change) > 1 or <-1 and adjusted p-value < 0.01. SDRF File E-GEAD-418.sdrf.txt Comment[AEExperimentType] RNA-seq of coding RNA Comment[BioProject] PRJDB10800 Comment[Last Update Date] 2021-09-10