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| Status |
Public on Sep 10, 2020 |
| Title |
bulk [NextFlex_NB_0319_003_S3] |
| Sample type |
SRA |
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| Source name |
brain
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| Organism |
Homo sapiens |
| Characteristics |
cell type: microglia
donor group: CTR+
donor id: 2018-064
brain bank: NetherlandsBrainBank
brain region: LPS
sequencing pool: 3
age: 103
gender: F
ph csf: 8.3
post mortem delay (h.mm): 9:50
sorter: NA
passedpreprocessingqc: YES
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| Extracted molecule |
total RNA |
| Extraction protocol |
Microglia were isolated by mechanical tissue dissociation and Percoll gradient centrifugation at 4°C followed by flow cytometry of viable (DAPI-, DRAQ5+), CD45+, CD11B+ cells.
All 25 RNA samples, with RIN values varying between 5.1 and 9.9, were enriched for poly(A)+ messenger RNA using NEXTflex Poly(A) Beads (BIOO Scientific, #NOVA-512980) according to the protocol in the manual. Fourteen µL of this mRNA-enriched poly(A)-tailed RNA was used as the input for the NEXTflex Rapid Directional qRNA-Seq kit (Bioo Scientific, #NOVA-5130-04). Library preparation was performed according to the manufacturers protocol. Quality and concentration of libraries from individual samples were assessed using the High Sensitivity dsDNA kit (Agilent, 067-4626) on a 2100 Bioanalyzer (Agilent) and a Qubit 2.0 Fluorometer (Life Technologies). Subsequently, individual libraries were combined into 3 sequencing pools with equal molar input per sample. 75bp paired-end sequencing was performed on an Illumina NextSeq 500 system. PhiX was added at 5% to both pools as an internal control before sequencing.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
NextSeq 550 |
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| Description |
bulk
mechanical
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| Data processing |
The NEXTflex barcode (9 base pairs) was stripeed from the sequence, using a custom bash script. The barcode was saved in a separate file for further use.
The sequencing reads were aligned with HISAT2 (version 2.1.0) to the GRCh38.92 reference genome with Ensembl annotation.
Further processing was done with samtools (version 1.9) and Picard Tools (version 1.140). This included sorting, assigning reads to a read group, verification of mate pair information and marking duplicates.
After these processing steps, reads were quantified using featureCounts (Subread version 1.6.2)
Reads from bulk samples were deduplicated using a bash script by BIOO Scientific (v2, date 11/1/14) , using the NEXTflex barcode that was saved in the additional file.
Genome_build: Homo.sapiens.GRCh38.92
Supplementary_files_format_and_content: A tab-delimited text file per sample containing gene counts used for bulk-sequencing (n=25).
Supplementary_files_format_and_content: UMItools_GEO.py: Demultiplexing and alignment pipeline.
Supplementary_files_format_and_content: readinCBC.csv contains the custom cell barcodes for Smartseq2 sequencing data.
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| Submission date |
Jun 25, 2020 |
| Last update date |
Nov 15, 2022 |
| Contact name |
Bart Eggen |
| E-mail(s) |
[email protected]
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| Phone |
+31503616432
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| Organization name |
UMCG Groningen
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| Department |
Department of Biomedical Sciences
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| Lab |
Section Molecular Neurobiology
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| Street address |
Antonius Deusinglaan 1
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| City |
Groningen |
| State/province |
Groningen |
| ZIP/Postal code |
9713AV |
| Country |
Netherlands |
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| Platform ID |
GPL21697 |
| Series (1) |
| GSE146639 |
Bulk and single cell sequencing (bc-Smart-seq2 and 10XGenomics) of human microglia from post mortem AD CNS tissue |
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| Relations |
| BioSample |
SAMN15368545 |
| SRA |
SRX8616689 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4636681_NextFlex_NB_0319_003_S3_sorted.bam.featureCounts.genes.txt.gz |
58.9 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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