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Sample GSM7947065 Query DataSets for GSM7947065
Status Public on Jul 29, 2024
Title OSN_ac1pCBSdel_rep2_RNA
Sample type SRA
 
Source name Olfactory epithilium
Organism Mus musculus
Characteristics tissue: Olfactory epithilium
genotype: Omp_GFP;ac1pCBSdel/del
Treatment protocol Mice were sacrificed using CO2 followed by cervical dislocation. The main olfactory epithelium (MOE) was dissected and transferred to ice-cold PBS. The MOE was cut in to small pieces with a razor blade, and then dissociated with a papain dissociation system (Worthington Biochemical). For RNA-seq, dissociated cells were washed once with sort media (PBS + 2% Fetal Bovine Serum) and then resuspended in sort media supplemented with 100 U/mL DNase I (Worthington Biochemical) and 500ng/mL DAPI (Invitrogen). For HiC samples, cells were fixed prior to sorting. For fixation, dissociated cells were resuspended in PBS + 1% methanol-free formaldehyde (Pierce). Cells were fixed at room temperature for 10 minutes, and then fixation was quenched by adding 1/10th volume of 1.25M glycine. Fixed cells were pelleted (500 rcf, 5 minutes, room temperature), washed once with PBS + 2% FBS, and then resuspended in sort media. All cells were passed through a 40uM cell strainer prior to sorting.
Growth protocol N/A
Extracted molecule total RNA
Extraction protocol For RNA-seq: RNA was isolated from tissue using TRIzol. Cell lysate was extracted with bromo-chloropropane and RNA was precipitated with 100% isopropanol supplemented with 10 μg of glycobluefor 10 min at room temperature and then pelleted at 16,000 x g for 30 min at 4°C. The RNA pellet was washed once with 75% ethanol and then resuspended in RNase-free water to a maximal concentration of 200 ng/μl. Genomic DNA contaminants were removed by Turbo DNase. Removal of Turbo DNase was performed by phenol:chloroform extraction and RNA was precipitated as described above and resuspended in RNase-free water and stored at -80°C. For in situ HiC was carried out according to Rao et al. 2014 and according to the manufacturer's instructions of the Arima HiC kit. For ChIP-seq: Cells were crosslinked with 1% formaldehyde for 10 minutes at room temperature. Formaldehyde was quenched by adding glycine to a final concentration of 0.125 M for 5 minutes at room temperature. Cells were then washed with 1X cold PBS with protein inhibitors twice and pelleted. Cell pellets were stored at -80°C till use. Cells were lysed in lysis buffer (50 mM Tris pH 7.5, 140 mM NaCl, 0.1% SDS, 0.1% sodium deoxycholate, 1% Triton X-100) for 10 minutes. Nuclei were spun for 10 minutes at 1000 x g and resuspended in the sonication buffer (10 mM Tris pH 7.5, 0.5% SDS) as 5million nuclei per 300 μl sonication buffer. Chromatin was sheared by Covaris (Peak power 105.0; Duty Factor 2.0; Cycle/Burst 200; Treatment 960 sec; Temperature 4-8°C). Following a spin at 13,000 x g for 10 minutes to remove debris, the sheared chromatin was diluted such that the final binding buffer concentration was 15 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% SDS) and incubated for 2 hours with dynabeads G pre-equilibrated in the binding buffer for pre-clearing of the chromatin. Post-cleared chromatin was then incubated with the specific antibody overnight (1 μg of antibody was used per 5 million nuclei). The next day, dynabeads G were added to the chromatin-antibody mix for 2 hours. A total of four washes with 1X wash buffer (100 mM Tris pH 7.5, 500 mM LiCl, 1% NP-40, 1% sodium deoxycholate) and one wash with TE buffer (10 mM Tris pH 7.5, 1 mM EDTA) were performed. The elution was performed at65°C for 1 hour in the elution buffer (1% SDS, 250 mM NaCl, 2 mM DTT). All steps, with the exception of the elution, were performed at 4°C. All buffers, with the exception of the TE and elution buffer contained 1X protease inhibitors. The eluted chromatin was reverse-crosslinked overnight at 65°C and the DNA was purified with AMPure XP beads (Beckman Coulter).
For RNA-Seq experiments: Libraries were made following the SMARTer Stranded Total RNA-Seq. Raw FASTQ files were aligned with either Tophat or STAR using mm10 reference genomes. The initial 4 bases of both paired reads were trimmed prior to alignment. For single cell RNA-seq: Libraries were generated using 10x Genomics 5' kits according to manufacturer's instructions. Raw FASTQ files were analyzed using cellranger. For in situ Hi-C experiments: Libraries were generated according to Rao et al. 2014 and the manufacturer's instructions of the Arima HiC kit. Raw FASTQ files were analyzed using HiC-Pro and hic files were generated (Servant et al. 2015). For WGBS: Libraries were constructed according to the Ultralow Methyl-Seq with TrueMethyl oxBS according to manufacturer's instructions. Analysis was performed using the instructions outlined in https://github.com/nugentechnologies/NuMetWG .
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model NextSeq 550
 
Description RNA-seq
Data processing Adapter Sequences were trimmed with cutadapt (v1.9.1) (--minimum-length 18)
Alignment: All samples were aligned to mm10. For paired-end data sets, only read 1 was used for alignment and downstream analysis. RNA-seq samples were aligned with STAR (v2.5.3a) with the following parameters: --outFilterIntronMotifs RemoveNoncanonical --outFilterType BySJout --outFilterMultimapNmax 20. HiC samples were aligned and processed using HiC-Pro (Servant et al. 2015 Genome Biology). 10x single cell RNA-seq was processed using cellranger.
Filtering: Unmapped reads and reads with a mapping quality below 30 were removed with Samtools (1.3.1).
For RNA-Seq, bigWig files were generated with RSeQC (2.6.4).
10x scRNAseq data were analyzed using 10x Genomics Cell Ranger 6.0.1 with a recovery of 4000-8000 cells per experiment. Data analysis was performed using Seurat​ and custom scripts.
Assembly: mm10
Supplementary files format and content: bigWig files at base pair resolution normalized to a library size of 10,000,000 reads
 
Submission date Dec 05, 2023
Last update date Jul 29, 2024
Contact name Lea Kiefer
E-mail(s) lea.kiefer@ucsf.edu
Organization name UCSF
Street address 1651 4th Street, Weill Neurosci RM 471A
City SAN FRANCISCO
State/province CA
ZIP/Postal code 94103
Country USA
 
Platform ID GPL21626
Series (1)
GSE249430 Cohesin trajectories shape the epigenetic and transcriptional landscape of clustered Protocadherin genes for single neuron identities
Relations
BioSample SAMN38688058
SRA SRX22793750

Supplementary file Size Download File type/resource
GSM7947065_OSN.aC1pCBSdel.small.Rep2.neg.bw 70.0 Mb (ftp)(http) BW
GSM7947065_OSN.aC1pCBSdel.small.Rep2.pos.bw 70.8 Mb (ftp)(http) BW
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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