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| Status |
Public on Jun 26, 2018 |
| Title |
s_PIMC_Rep1 |
| Sample type |
SRA |
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| Source name |
Stable plaque inner mass cells
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| Organism |
Homo sapiens |
| Characteristics |
tissue: Cells derived from inner mass of stable carotid atherosclerotic plaque
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| Treatment protocol |
Plaque tissue samples with average size 12±1×4±1×6±1 millimeters and weight 300-500 mg were treated two times with at least 2 volumes (volume : weight) of lysis buffer (0,5% NP-40, 2 mM EDTA, 0,01 M Tris-HCl, pH=7,5) for 10 minutes at the room temperature. Then tissue samples were washed 5 times with PBS, minced finely into small pieces (1 mm3) by sterile scalpels with subsequent incubation in 0,2 % Collagenase type II (prepared in IMDM from 1 % collagenase II (Invitrogen) in Hanks’ solution (Sigma-Aldrich) for 24 hours at 37 °С, 5 % CO2. Hydrolyzed plaque fragments were centrifuged 10 min at 400 g, resuspended in 6 ml IMDM, 10 % FBS, 100 μg/ml penicillin and streptomycin (Invitrogen, Carlsbad, CA, USA) (IMDM-FBS), placed in the 10-mm tissue culture dishes and cultivated at 37 °C, 5 % CO2. After 3 days adherent PIMC were washed 3 times with IMDM to remove tissue fragments, fresh IMDM-FBS was added and cells were cultivated at 37 °C, 5 % CO2 with a medium change every 5 days for 20-25 days until about 80 % of confluent cell monolayer was formed. Cells were subcultured with the solution of 0.2 % collagenase type II in IMDM (30-40 minutes treatment at 37°С, with subsequent addition of 4 volumes of IMDM-FBS with 0,5 % gelatin).
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
The first passage PIMC from two stable and two vulnerable atheromes were washed with PBS twice and detached from Petri dishes using 0.2 % collagenase type II in IMDM, centrifuged 10 min at 400 g, then 750 µL of Trizol reagent was added to the pellet and frozen at 80°C until RNA extraction. PIMCs in Trizol were thawed, mixed with 0.15 mL chloroform, vortexed for 15 s, and incubated at ambient temperature for 2 min. Thereafter, samples were centrifuged at 12,000g for 15 min at 4°C. Aqueous phase was transferred to a fresh tube and precipitated after adding 20 µg glycogen and 375 µL isopropanol. RNA samples were treated with RNase-free DNase I (Fermentas, Vilnius, Lithuania) and precipitated in 70 % ethanol, 0,3 М NaAc, 20 µg glycogen. The concentration of extracted RNA and RNA integrity number (RIN) and were tested using NanoDrop 2000 (Thermo Fisher Scientific, Wilmington, DE, USA) and Agilent RNA 6000 Pico Kit on Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA).
The libraries were prepared for the sequencing using Illumina TruSeq Stranded mRNA Sample Preparation Kit and were sequenced with the HiSeq 2500.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
RNA-seq reads were quality-filtered using FASTQ Quality Filter (-q 20 –p 75) from FASTX-toolkit (http://hannonlab.cshl.edu/fastx_toolkit/).
Ribosomal RNA reads were filtered out using SortMeRNA tool.
RNA-seq reads were aligned on GRCh38 (Ensembl release 91) using HISAT2 aligner (--score-min L,0,-0.5)
FeatureCounts tool from the Subread package was used to count reads to genomic features
Genome_build: GRCh38 (Ensembl release 91)
Supplementary_files_format_and_content: tabular files were generated with featureCounts and contain raw gene counts
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| Submission date |
Jun 25, 2018 |
| Last update date |
Jun 26, 2018 |
| Contact name |
Petr Laktionov |
| E-mail(s) |
laktionov@mcb.nsc.ru
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| Organization name |
Institute of molecular and cellular biology SD RAS
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| Lab |
Genomics lab
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| Street address |
Lavrenitev ave 8/2
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| City |
Novosibirsk |
| ZIP/Postal code |
630090 |
| Country |
Russia |
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| Platform ID |
GPL16791 |
| Series (1) |
| GSE116243 |
Isolation and culture of carotid atherosclerotic plaque inner mass cells |
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| Relations |
| BioSample |
SAMN09483726 |
| SRA |
SRX4295871 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3215457_s_PIMC_Rep1_counts.tabular.xlsx |
7.8 Mb |
(ftp)(http) |
XLSX |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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