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| Status |
Public on Jun 01, 2021 |
| Title |
1006_ovary |
| Sample type |
SRA |
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| Source name |
ovary
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| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
organ: ovary
age: 16.5
Sex: Female
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| Extracted molecule |
cytoplasmic RNA |
| Extraction protocol |
Organ samples of approximately 10 mm3 were bluntly dissected and then homogenized in 0.5-1mL of lysis buffer (150 mM NaCl, 50 mM HEPES pH 7.4, 25 ug/mL digitonin with 1000 U/mL SUPERase-In Rnase inhibitor added just prior to application) to lyse the cytoplasmic membrane. The mixture was lightly homogenized in a dounce homogenizer with an autoclaved B pestle, incubated on ice for 5 minutes and then centrifuged for 2 min at 1000 rpm at 4oC. Supernatant, containing the cytoplasmic fraction, was mixed with pre-chilled 7.5 mL of Trizol and 1.5 mL of chloroform and then centrifuged for 35 min at 4000 rpm at 4oC. The aqueous portion was transferred to 4.5 mL of chilled chloroform, mixed and centrifuged for 10 min at 4000 rpm at 4oC. The resulting aqueous portion was precipitated with 4.5 mL of isopropanol overnight in 80oC, centrifuged for 45 min at 4oC at 4000 rpms, washed with 10 mL of ethanol and re-suspended in RNAse-free water.
Cytoplasmic RNA samples were submitted to BGI Genomics for selection of polyadenylated RNAs, and strand-specific, paired-end library preparation. During this study, we used two methods of RNA sequencing with the same read depth: BGI DNA nanoball sequencing (DNBseq), which produces 150 bp read lengths, and Illumina sequencing, which produces 100 bp read lengths. For samples sequenced using Illumina sequencing, samples were pooled in groups of 5-7 and applied to a single lane of an Illumina HiSeq 2500/4000 instrument.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
DNBSEQ-G400 |
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| Data processing |
We used Bowtie1 to concordantly map read pairs (-X 600) with a single preferred mapping location (-m 1) with the tryhard switch (-y) that forces it to exhaustively search each read pair against the reference genome. The same alignment parameters were used in this study with either mm10 or rn6 genomes.
The generated bam file was strand separated and then intersected with the same-oriented annotation for full-length L1s. Manual curation of resulting L1 loci with greater than 10 mapped reads was performed using IGV
After aligning the .fastq sequences to the appropriate reference genome (mm10 or rn6), we used a list of LINE-1 element genomic coordinates downloaded from L1 base (http://l1base.charite.de/) to quantify how many reads from the aligned files overlapped with annotated LINE-1 element coordinates. This was performed using 'bedtools coverage'. L1 loci are annotated at 'UID-' and their corresponding number of reads are in the 10th column from the left side of the .txt file.
L1Base - L1Resources<http://l1base.charite.de/>
L1Resources. This web portal is dedicated to putatively active LINE-1 elements. What are LINE-1s? LINE-1 (Long Interspersed Nucleotide Element 1, L1) are the only autonomous retrotransposons in the group of non-LTR retrotransposons.
All scripts and protocols used in this analysis are available in our previous publication RNA Next-Generation Sequencing and a Bioinformatics Pipeline to Identify Expressed LINE-1s at the Locus-Specific Level (doi: 10.3791/59771).
Genome_build: mm10 or rn6
Supplementary_files_format_and_content: L1 loci are annotated at 'UID-' and their corresponding number of reads are in the 10th column from the left side of the .txt file.
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| Submission date |
Sep 30, 2020 |
| Last update date |
Jun 01, 2021 |
| Contact name |
Victoria Belancio |
| E-mail(s) |
vperepe@tulane.edu
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| Organization name |
Tulane University School of Medicine
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| Department |
Structural and Cellular Biology
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| Street address |
1430 Tulane Avenue
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| City |
New Orleans |
| State/province |
LA |
| ZIP/Postal code |
70112 |
| Country |
USA |
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| Platform ID |
GPL28457 |
| Series (1) |
| GSE158831 |
Organ-, sex-, and age-dependent patterns of endogenous LINE-1 mRNA expression at a single locus resolution |
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| Relations |
| BioSample |
SAMN16315965 |
| SRA |
SRX9220893 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4811650_1006-ovary_rmdup_mm10_sorted_bowtie_tryhard_minus_bottom.txt.gz |
211.3 Kb |
(ftp)(http) |
TXT |
| GSM4811650_1006-ovary_rmdup_mm10_sorted_bowtie_tryhard_plus_top.txt.gz |
190.2 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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