|
| Status |
Public on Mar 16, 2020 |
| Title |
Agrp_neurons_RNA_seq_Phf6_cKO-5_Fed_condition |
| Sample type |
SRA |
| |
|
| Source name |
Agrp neurons
|
| Organism |
Mus musculus |
| Characteristics |
cell type: Agrp neurons
strain: C57BL/6
age: P25-P30
genotype: Phf6 cKO
feeding condition: Fed
|
| Treatment protocol |
For mice in overnight fasted condition, food deprivation was started from 5 pm the day before experiment, and only water was provided.
|
| Growth protocol |
AGRP neurons were obtained by sorting tdTomato+ red fluorescent neurons from the hypothalamus of P25-P30 Phf6cKO mice (Phf6 flox/y; AgRP-Cre/+; Ai9/+) and Phf6WT mice (AgRP-Cre/+; Ai9/+) littermate mice.
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
Mice were sacrificed and around 1 mm3 hypothalamic tissue from each mouse was obtained with 1 mm-diatmeter micro punch in ice-cold dissection solution (1xHBSS with 10 mM HEPES and 23 mM D-glucose). Cell dissociation was conducted using Papain Dissociation System kit (Worthington-Biochem: #LK003150), and the cell dissociation procedure was conducted under the guidance of the kit.
1000 tdTomato+ AgRP neurons in 10 ml RNAseq lysis buffer with RNase inhibitor per sample were submitted for cDNA synthesis by using SMART-seq v4 Ultra Low Input RNA kit (Clontech). First-strand cDNA synthesis was primed by the 3’ SMART-Seq CDS Primer II A and used the SMART-Seq v4 oligonucleotide for template switching at the 5’ end of the transcript. cDNAs were amplified by LD PCR. PCR II A amplified cDNA from the SMART sequences introduced by 3’ SMART-Seq CDS Primer II A and the SMART-Seq v4 oligonucleotide for 15 cycles. PCR-amplified cDNA was purified by immobilization on AMPure XP beads. The beads were then washed with 80% ethonal and cDNA is eluted with elution buffer. Amplified cDNAs were then validated using the Agilent 2100 Bioanalyzer and Agilent’s High Sensitivity DNA kit. 5 ng full-length cDNA output for next-generation sequencing was processed with the Nextera XT DNA library preparation kit (Illumina). Libraries were sequenced on the Illumina HiSeq X Ten.
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| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
HiSeq X Ten |
| |
|
| Data processing |
RNA-seq Reads were trimmed by Trimmomatic (version 0.36) and then aligned to the mouse reference genome (GRCm38/mm10, downloaded from UCSC, http://genome.ucsc.edu/) using Hisat2 (version 2.1.0).
Next, raw counts of genes were quantified using featureCounts from Subread package (version 1.6.2). Normalized counts were obtained by normalizing raw counts with size factors using the "median ratio method" in DESeq2 1.20.0 package.
Subsequent differentiated expressed gene analysis was performed using DESeq2 with default settings.
Genome_build: mm10
Supplementary_files_format_and_content: csv files containing raw counts and normalized counts obtained from DESeq2 package
|
| |
|
| Submission date |
Oct 29, 2019 |
| Last update date |
Mar 16, 2020 |
| Contact name |
Qian Li |
| E-mail(s) |
liqian@shsmu.edu.cn
|
| Organization name |
Shanghai Jiao Tong University School of Medicine
|
| Department |
Department of Anatomy and Physiology
|
| Street address |
280 South Chongqing Road, Building No.5, Room 408
|
| City |
Shanghai |
| ZIP/Postal code |
200025 |
| Country |
China |
| |
|
| Platform ID |
GPL21273 |
| Series (2) |
| GSE139543 |
Chromatin binding protein PHF6 regulates activity-dependent transcription networks to promote hunger response [RNA-Seq] |
| GSE139545 |
Chromatin binding protein PHF6 regulates activity-dependent transcription networks to promote hunger response |
|
| Relations |
| BioSample |
SAMN13152635 |
| SRA |
SRX7069128 |
