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Sample GSM5452260 Query DataSets for GSM5452260
Status Public on Jul 12, 2022
Title PAM-1 ChIRP-seq input
Sample type SRA
 
Source name ASC
Organism Mus musculus
Characteristics cell type: ASC
strain: Tg:Pax7-nGFP
antibody: none
Treatment protocol none
Growth protocol Hindlimb skeletal muscles from Tg:Pax7-nGFP mice were dissected and minced, followed by digestion with Collagenase II (LS004177, Worthington, 1000 units/mL) for 90 min at 37°C in water bath shaker. Digested muscles were then washed in washing medium (Ham’s F-10 medium (N6635, Sigma) containing 10% heat inactivated horse serum (Gibco, 26050088) with 1% penicillin/ streptomycin, followed by incubating in digestion medium with Collagenase II (100 units/mL) and Dispase (1.1 unit/mL, Gibco, 17105-041) for additional 30 min. Suspensions were then passed through 20G syringe needle to release myofiber-associated SCs. Mononuclear cells were filtered with a 40mm cell strainer, followed by cell sorting using BD FACSAria Fusion Cell Sorter (BD Biosciences). BD FACSDiva (BD Biosciences, version 8.0.1) software was used to manage machine startup, data acquisition and analysis of flow cytometry data. Culture dish were coated with poly-D-lysine (Sigma, P0899) and Matrigel (BD Bioscience, 356234). FACS isolated SCs were seeded in coated culture dish and cultured in Ham’s F10 medium with 10% heat inactivated horse serum, 5ng/mL FGF-Basic (AA 10-155) (Gibco, PHG0026).
Extracted molecule genomic DNA
Extraction protocol 10 million cells were collected per ChIRP experiment for separated odd and even probe pools. Cell pellets were cross-linked with 1% Glutaraldehyde in 40mL PBS on rotator for 10 minutes at room temperature, followed by quenching the cross-linking reaction with 2mL 1.25M Glycine for 5 minutes and resuspend in 1mL chilled PBS. Cell pellets were collected at 2000RCF for 5 minutes at 4 °C, followed by removing PBS, snap frozen with liquid nitrogen and stored at -80°C. Cell pellets were lysed and sonicated using Covaris S220 sonicator, then aliquoted into two 1mL samples. Before ChIRP experiment, DNA were extracted for quality control with size ranging from 100-500bp. For ChIRP experiment, 10mL of lysate were saved for DNA input. 1mL of sonicated lysate was mixed with 2mL of hybridization buffer (750mM NaCl, 50mM Tris-HCl pH7, 1mM EDTA, 1% SDS, 15% Formamide, 1x protease inhibitor and 1x RNase inhibitor). 100pmol of odd and even ChIRP probes were added separately to the hybridization mixture and incubate at 37°C for 4 hours with rotation. After the hybridization was completed, 100uL of streptavidin magnetic C1 beads (Life Technologies, 65001) were washed thrice with hybridization buffer and added to each ChIRP reaction for extra 30 minutes incubation at 37°C with rotation. After the hybridization completed, 1mL of wash buffer (2X SSC, 0.5% SDS and 1x protase inhibitor) was used to wash the beads for 5 times using magnetic stand. Input control and PAM-1 bound DNA was eluted, followed by library construction.
NEBNext Ultra II DNA Library Preparation kit for Illumina (NEB)
 
Library strategy OTHER
Library source genomic
Library selection other
Instrument model Illumina HiSeq 1500
 
Data processing Library strategy: ChIRP-seq
The raw data were first preprocessed by initial quality assessment, adaptors trimming, and low-quality filtering
The remaining reads were aligned to the mouse reference genome (mm9) by Bowtie2
The binding sites were identified by software MACS2
Genome_build: mm9
 
Submission date Jul 14, 2021
Last update date Jul 12, 2022
Contact name Yile Huang
E-mail(s) huangyilekuaile@gmail.com
Phone 56289467
Organization name The Chinese University of HongKong
Department Chemical Pathology
Street address Room 503, Li Ka Shing Health and Science Buiding, Prince of Wales Hospital, Sha Tin
City Sha Tin
State/province Hong Kong
ZIP/Postal code 999077
Country China
 
Platform ID GPL18480
Series (2)
GSE180071 seRNA PAM-1 interacts with Ddx5 to regulate skeletal muscle satellite cell activation and aging through trans regulation of Timp2 expression [PAM-1 ChIRP-seq]
GSE180073 seRNA PAM-1 interacts with Ddx5 to regulate skeletal muscle satellite cell activation and aging through trans regulation of Timp2 expression
Relations
BioSample SAMN20207701
SRA SRX11439173

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data not applicable for this record

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