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| Status |
Public on Jul 29, 2024 |
| Title |
OSN_WT_rep2_RNA |
| Sample type |
SRA |
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| Source name |
Olfactory epithilium
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| Organism |
Mus musculus |
| Characteristics |
tissue: Olfactory epithilium
genotype: Omp_GFP
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| 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.
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| Growth protocol |
N/A
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| 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 .
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
NextSeq 550 |
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| Description |
RNA-seq
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| 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
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| Submission date |
Dec 05, 2023 |
| Last update date |
Jul 29, 2024 |
| Contact name |
Lea Kiefer |
| E-mail(s) |
lea.kiefer@ucsf.edu
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| Organization name |
UCSF
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| Street address |
1651 4th Street, Weill Neurosci RM 471A
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| City |
SAN FRANCISCO |
| State/province |
CA |
| ZIP/Postal code |
94103 |
| Country |
USA |
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| Platform ID |
GPL21626 |
| Series (1) |
| GSE249430 |
Cohesin trajectories shape the epigenetic and transcriptional landscape of clustered Protocadherin genes for single neuron identities |
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| Relations |
| BioSample |
SAMN38688070 |
| SRA |
SRX22793739 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7947053_OSN_WT_RNASeq.Rep2.neg.bw |
128.4 Mb |
(ftp)(http) |
BW |
| GSM7947053_OSN_WT_RNASeq.Rep2.pos.bw |
130.0 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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