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| Status |
Public on Apr 15, 2019 |
| Title |
HEK_a9eCBS_TotRNASeq_rep2 |
| Sample type |
SRA |
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| Source name |
HEK293T cells transfected with dCas9 targeting the eCBS of Pcdha9
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| Organism |
Homo sapiens |
| Characteristics |
tissue: HEK293T cells
extract_protocol: total RNA
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| Growth protocol |
SK-N-SH cells were purchased from ATCC and cultured in RPMI-1640 supplemented with 10% (vol/vol) FBS, 1X GlutaMax, 1mM sodium pyruvate, 1X non-essential amino acids, 1% penicillin-streptomycin. HEK293T cells were purchased from ATCC and cultured in DMEM supplemented with 10% (vol/vol) FBS, 1X GlutaMax, 1mM sodium pyruvate, 1X non-essential amino acids, 1% penicillin-streptomycin. Cells were maintained at 37°C in a 5% (vol/vol) CO2 incubator.
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| Extracted molecule |
total RNA |
| Extraction 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), 4mM MgCl2, and 500ng/mL DAPI (Invitrogen). For Crosslinked-ChIP 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 5 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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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
NextSeq 550 |
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| Description |
captureRNA-Seq, Paired End
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| Data processing |
For RNA-Seq experiments, raw FASTQ files were aligned with either Tophat or STAR using hg19 or mm10 reference genomes. When libraries were made following the SMARTer Stranded Total RNA-Seq, the initial 4 base pairs of both paired reads were trimmed prior to alignment.
For ChiP-Seq experiments, raw FASTQ files were aligned using Bowtie2 using hg19 reference genome upon adapter sequences removal using CutAdapt. Uniquely aligning reads were selected using Samtools and reads with alignment quality below 30 (-q 30) were removed. The HOMER software package was used to generate signal tracks.
For in situ Hi-C experiments, raw FASTQ files were processed through use of the Juicer Tools Version 1.76 pipeline (Durand et al., 2016) with one modification. Reads were aligned to hg38 using BWA 0.7.17 mem algorithm and specifying the -5 option implemented specifically for in situ Hi-C data.
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. Crosslinked ChIP-Seq samples were aligned with Bowtie2 (2.3.2) with default parameters. RNA-seq samples were aligned with STAR (v2.5.3a) with the following parameters: --outFilterIntronMotifs RemoveNoncanonical --outFilterType BySJout --outFilterMultimapNmax 20
Filtering: Unmapped reads and reads with a mapping quality below 30 were removed with Samtools (1.3.1). For ChIP-seq, duplicate reads were marked with Picard and removed with samtools.
For ChIP-Seq, bigWig files were generated using HOMER (4.7) (-fsize 1e20 -fragLength given). For RNA-Seq, bigWig files were generated with RSeQC (2.6.4).
Genome_build: 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 |
Jun 15, 2018 |
| Last update date |
Apr 15, 2019 |
| Contact name |
Daniele Canzio |
| Organization name |
University of California San Francisco
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| Department |
Neurology
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| Street address |
555 Mission Bay Boulevard South
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| City |
San Francisco |
| State/province |
CA |
| ZIP/Postal code |
94158 |
| Country |
USA |
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| Platform ID |
GPL21697 |
| Series (1) |
| GSE115862 |
Antisense lncRNA transcription mediates DNA demethylation to drive stochastic Protocadherin α promoter choice |
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| Relations |
| BioSample |
SAMN09430239 |
| SRA |
SRX4221447 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3192028_HEK_a9eCBS_TotRNA_rep2.neg.bw |
13.6 Mb |
(ftp)(http) |
BW |
| GSM3192028_HEK_a9eCBS_TotRNA_rep2.pos.bw |
14.4 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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