|
| Status |
Public on Feb 26, 2021 |
| Title |
lepr-expressing POMC pulldown #3 |
| Sample type |
SRA |
| |
|
| Source name |
arcuate nucleus (ARC)
|
| Organism |
Mus musculus |
| Characteristics |
genotype: POMCDre+/- LeprCre+/- ROSA26lSlrSrEGFPL10a+/-
Sex: pooled male and female
tissue: hypothalami
feeding: Random-fed
|
| Treatment protocol |
All mice were rapidly decapitated and tissue was taken between 11-13h
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Samples were homogenized in Trizol LS, extracted with Chloroform, precipitated with isopropranol, washed with 80% Ethanol, resuspende din Nuclease free water and treated with DNAse I. The TRAP technique was performed using a modified version of (Heiman et al., 2008) on hypothalami of mice described under “Study design of bacTRAP (EGFPL10a) mice”. Concentration was analyzed using QuBit/Bioanalyzer and remaining samples were stored at -80°C until sequencing.
Total RNA was used for first strand cDNA synthesis, using both poly(T) and random primers, followed by second strand synthesis and isothermal strand-displacement amplification. For library preparation, the Illumina Nextera XT DNA sample preparation protocol was used, with 1 ng cDNA input. After validation (Agilent 2200 TapeStation) and quantification (Invitrogen Qubit System) all 24 transcriptome libraries were pooled. The pool was quantified using the Peqlab KAPA Library Quantification Kit and the Applied Biosystems 7900HT Sequence Detection and sequenced on a Illumna NovaSeq S2 flowcell with a PE100 protocol. The RNA sequencing pipeline utilizes the GRCm38 assembly of the mouse genome as gene sets from Ensembl release 96 (Yates et al., 2015).
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Description |
3 males and 3 females pooled
|
| Data processing |
Illumina TruSeq mRNA stranded protocol and the HiSeq 4000 sequencer with a PE75 read length run
Data was processed using the nf-core/rnaseq v1.4.2 pipeline
Alignment to reference genome was done using STAR vSTAR_2.6.1d
Gene abundance estimation was done using Salmon 0.14.1
Differential gene expression analysis of IP/Input normalised gene counts was carried out using DESeq2 1.26.0
Genome_build: GRCm38.p6 (Genome Reference Consortium Mouse Reference 38), INSDC Assembly GCA_000001635.8, Jan 2012
Supplementary_files_format_and_content: Excel sheet of DESeq2 contrasts glp1r and lepr
|
| |
|
| Submission date |
Jul 03, 2020 |
| Last update date |
Feb 26, 2021 |
| Contact name |
Nasim Biglari |
| E-mail(s) |
nasim.biglari@sf.mpg.de
|
| Organization name |
Max Planck Institute for Metabolism Research
|
| Street address |
Gleueler Str. 50
|
| City |
Cologne |
| ZIP/Postal code |
50931 |
| Country |
Germany |
| |
|
| Platform ID |
GPL24247 |
| Series (1) |
| GSE153753 |
Functionally Distinct Pomc-Expressing Neuron Subpopulations in Hypothalamus Revealed by Intersectional Targeting |
|
| Relations |
| BioSample |
SAMN15438398 |
| SRA |
SRX8664652 |
