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| Status |
Public on Sep 22, 2023 |
| Title |
391Pos |
| Sample type |
SRA |
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| Source name |
Hippocampus
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| Organism |
Mus musculus |
| Characteristics |
tissue: Hippocampus
cell type: Neuron
genotype: Camk2a-Cre/ERT2+;NuTRAP+
fraction: Positive
library prep_kit: NEBNext Ultra II Directional RNA Library Prep Kit for Illumina
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| Extracted molecule |
total RNA |
| Extraction protocol |
RNA from the hippocampus of one hemisphere of Tam-induced Camk2a-cre/ERT2+;NuTRAP+ mice was isolated via the TRAP method (Input, Negative, and Positive fractions). Tissue was minced into small pieces and homogenized in 100 ul ice-cold homogenization buffer (50 mM Tris, pH 7.4; 12 mM MgCl2; 100 mM KCl; 1% NP-40; 1 mg/mL sodium heparin; 1 mM DTT) supplemented with 100 ug/mL cycloheximide (#C4859-1ML, Millipore Sigma), 200 units/mL RNaseOUT™ Recombinant Ribonuclease Inhibitor (#10777019; Thermofisher), and 1X cOmplete™, EDTA-free Protease Inhibitor Cocktail (#11836170001; Millipore Sigma) with a Kimble pellet pestle cordless motor with one pulse of 10 sec/each on ice. An additional 300 uL of homogenization buffer was added and homogenized with pellet pestle cordless motor with one pulse of 10 sec/each (on ice). Volume was taken up to 1.5 mL with homogenization buffer and centrifuged at 12,000 x g for 10 minutes at 4°C. After centrifugation, 100 uL of the supernatant was saved as input. The remaining supernatant was transferred to a 2 mL round-bottom tube and incubated with 5 ug/uL of anti-GFP antibody (ab290; Abcam) at 4°C with end-over-end rotation for one hour. Dynabeads™ Protein G for Immunoprecipitation (#10003D; Thermofisher) were washed three times in 1 mL ice-cold low-salt wash buffer (50mM Tris, pH 7.5; 12mM MgCl2; 100mM KCl; 1% NP-40; 100μg/ml cycloheximide; 1mM DTT). After removal of the last wash, the homogenate/antibody mixture was transferred to the 2 mL round-bottom tube containing the washed Protein-G Dynabeads and incubated at 4°C with end-over-end rotation for an additional two hours. Magnetic beads were collected using a DynaMag-2 magnet and the unbound- ribosomes and associated RNA saved as the “negative” fraction. Beads were then washed three times with 1 mL of high-salt wash buffer (50mM Tris, pH 7.5; 12mM MgCl2; 300mM KCl; 1% NP-40; 100μg/ml cycloheximide; 2mM DTT). Following the last wash, 350 uL of Buffer RLT (Qiagen) supplemented with 3.5 uL 2-β mercaptoethanol was added directly to the beads and incubated with mixing on a ThermoMixer (Eppendorf) for 10 minutes at room temperature. The beads were magnetically separated and the supernatant containing the target bead-bound ribosomes and associated RNA was transferred to a new tube. 350 uL of 100% ethanol was added to the tube (“positive” fraction) and then loaded onto an RNeasy MinElute column. RNA was isolated using RNeasy Mini Kit (#74104, Qiagen), according to manufacturer’s instructions. RNA was quantified with a Nanodrop 2000c spectrophotometer (Thermofisher Scientific) and its quality assessed by HSRNA screentape with a 2200 Tapestation analyzer (Agilent Technologies)
The NEBNext Ultra II Directional Library Prep Kit for Illumina (#NEBE7760L; New England Biolabs Inc., Ipswich, MA) was used on 100 ng of total RNA for the preparation of strand-specific sequencing libraries from each TRAP-isolated RNA sample (input, negative fraction, and positive fraction), according to manufacturer's instructions. Briefly, polyA containing mRNA was purified using oligo-dT attached magnetic beads. mRNA was chemically fragmented and cDNA synthesized. For strand-specificity, the incorporation of dUTP instead of dTTP in the second strand cDNA synthesis does not allow amplification past this dUTP with the polymerase. Following cDNA synthesis, each product underwent end repair process, the addition of a single ‘A’ base, and finally ligation of adapters. The cDNA products were further purified and enriched using PCR to make the final library for sequencing. Library sizing was performed with HS DNA ScreenTape (#5067-5582; Agilent Technologies) and libraries were quantified by Qubit dsDNA HS Assay Kit (ThermoFisher Scientific). The libraries for each sample were pooled at 4 nM concentration and sequenced using an Illumina NextSeq 6000 system (PE75bp) at the Oklahoma Medical Research Foundation Genomics Facility.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
NextSeq 550 |
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| Data processing |
Following sequencing, reads were trimmed, aligned, differential expression statistics and correlation analyses were performed in Strand NGS software package (Agilent)
Reads were aligned against the Mm10 build of the mouse genome (2014.11.26). Alignment and filtering criteria included: adapter trimming, fixed 2bp trim from 5’ and 6bp from 3’ ends, a maximum number of one novel splice allowed per read, a minimum of 90% identity with the reference sequence, a maximum of 5% gap, trimming of 3’ end with Q<30.
Alignment was performed directionally with Read 1 aligned in reverse and Read 2 in forward orientation. Reads were filtered based on the mapping status and only those reads that aligned normally (in the appropriate direction) were retained.
Normalization was performed with the DESeq algorithm
Transcripts with an average read count value >20 in at least 100% of the samples in at least one group were considered expressed at a level sufficient for quantitation per tissue and those transcripts below this level were considered not detected/not expressed and excluded, as these low levels of reads are close to background and are highly variable.
For statistical analysis of differential expression, a one-way ANOVA with the factor of TRAP fraction and a Benjamini-Hochberg Multiple Testing Correction followed by Student-Newman Keuls post hoc test were used. For those transcripts meeting this statistical criterion, a fold change >|1.25| cutoff was used to eliminate those genes which were statistically significant but unlikely to be biologically significant and orthogonally confirmable due to their very small magnitude of change.
Assembly: mm10
Supplementary files format and content: Tab-delimited text file contains RPKM values for each sample.
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| Submission date |
Mar 23, 2023 |
| Last update date |
Sep 22, 2023 |
| Contact name |
Kyla Tooley |
| E-mail(s) |
kyla-tooley@omrf.org
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| Organization name |
Oklahoma Medical Research Foundation
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| Department |
Genes and Human Disease
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| Lab |
Dr. Willard Freeman
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| Street address |
825 NE 13th St
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| City |
Oklahoma City |
| State/province |
OK |
| ZIP/Postal code |
73104 |
| Country |
USA |
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| Platform ID |
GPL21626 |
| Series (2) |
| GSE228043 |
Differential usage of DNA modifications in neurons, astrocytes and microglia [RNA-seq] |
| GSE228045 |
Differential usage of DNA modifications in neurons, astrocytes and microglia |
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| Relations |
| BioSample |
SAMN33870920 |
| SRA |
SRX19757012 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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