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Sample GSM4036785

Query DataSets for GSM4036785

Status

Public on Aug 16, 2019

Title

AC3174 treated sample 3 ip DVC [G06_S6]

Sample type

SRA

Source name

AC3174 treated ip DVC

Organism

Mus musculus

Characteristics

strain: C57/Bl6
age: 9 weeks
Sex: male
treatment: AC3174
brain site/tissue: mouse dorsovagal complex (DVC)
ip antibody: anti-pS6 240/244 antibody (#2215, Cell Signalling Technology)
rna sample type: ip
molecule subtype: ribosomal RNA

Treatment protocol

Mice were randomized into treatment groups and received an ip injection (10 µL/g of body weight) of either saline , AC710222 (2.5 or 10 µg/kg), exenatide (1 µg/kg, n=8), AC3174 (1 µg/kg), AC170222 (2.5 µg/kg) or a combination of these drugs

Growth protocol

9 weeks old C57/Bl6 male mice were maintained on standard chow and fasted for 6h before treatment administration

Extracted molecule

total RNA

Extraction protocol

Samples were homogenised in a homogenisation buffer containing 10 mM HEPES (pH 7.4), 150 mM KCl, 5 mM MgCl2, 100 nM calyculin A, 2 mM DTT, 100 U/mL RNasin, 100 mg/mL cycloheximide and Roche protease and phosphatase inhibitor cocktails and clarified by centrifugation at 2,000g at 4 C for 10 min. Supernatants were resuspended in 1 ml of 10% NP40 and 10 L of 1,2-diheptanoyl-sn-glycero-3-phosphocholine (DHPC) and centrifuged at 16,100 g at 4 C for 10 min. 25 L of the sample was used for RNA extraction (input sample) using Qiagen RNeasy Micro kit. The rest of the supernatant was used for ribosome immunoprecipitation by incubation with an antibody-beads conjugate containing anti-pS6 240/244 antibody (#2215, Cell Signalling Technology) and protein A dynabeads resuspended in the homogenization buffer supplemented with 50 L of 10% NP40 and 10 L of DHPC for 1 mL of buffer C, and 1 M ZK10 for 10 min at 4 C. The beads were washed twice with wash buffer (10 mM HEPES, 350 mM KCl, 5 mM MgCl2, 2 mM DTT, 1% NP40, 100 U/mL RNasin, 100 mg/mL cycloheximide, and Roche protease and phosphatase inhibitor cocktails) and resuspended in RLT for subsequent RNA extraction using Qiagen RNeasy Micro kit.
RNA was amplified using Clontech Smartseq V4 Ultra Low-input RNA kit and the amplified cDNA was used for library preparation with Illumina Nextera XT kit.

Library strategy

RIP-Seq

Library source

transcriptomic

Library selection

other

Instrument model

Illumina NextSeq 500

Description

G06_S6

Data processing

Fastq reads were mapped to the Mouse GRCm38 genome using Tophat V2.0.11
Gene counts were determined using htseq-count 0.6.1p1.
Genome_build: GRCm38
Supplementary_files_format_and_content: raw_count.tsv contain raw gene-level counts in tab-delimited text

Submission date

Aug 15, 2019

Last update date

Aug 16, 2019

Contact name

Brian Yee Hong Lam

E-mail(s)

yhbl2@cam.ac.uk

Organization name

University of Cambridge

Department

Metabolic Research Laboratories