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| Status |
Public on Sep 10, 2020 |
| Title |
single_cell_barcoded_SmartSeq2 [14-3] |
| 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: SP8
age: 103
gender: F
ph csf: 8.3
post mortem delay (h.mm): 9.5
sorter: XDP
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 5.7 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 2 sequencing pools of 13 samples each with equal molar input. 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.
single cell RNA-Seq, bc-Smart-seq2 :Microglia were isolated by mechanical tissue dissociation and Percoll gradient centrifugation at 4°C followed by flow cytometry of single, viable (DAPI-, DRAQ5+), CD45+, CD11B+ cells.
single cell RNA-Seq, bc-Smart-seq2 : After cell lysis and barcoded poly-dT primer annealing, RNA was reverse-transcribed with a biotinylated barcoded template switching oligo (BC-TSO). Primer-dimers and small fragments were removed by exonuclease treatment. cDNA libraries were amplified and multiplexed. When cDNA libraries showed an average size of 1.5-2 kb, libraries were tagmented and indexed according to the Illumina Nextera XT DNA preparation kit (Illumina, FC-131-1024) with an input of 500 pg cDNA. Samples were sequence on an Ilumina NextSeq 500. using 75 bp paired-end reads. Read 1 comprises the poolbarcode and read 2 the cDNA.
10X Genomics: 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.
10X Genomics: The single-cell barcoded libraries were constructed according to the instructions of the Single Cell 3’ Reagent Kits v2 (10x Genomics). Briefly, cells were loaded into a slot of a Chromium chip and GEMs were incubated in a thermal cycler to generate barcoded cDNA. After amplification, the cDNA was fragmented and processed for sequencing by ligating adapters and sample indices. The libraries were sequenced on an Illumina NextSeq 500 system with a sequencing depth up to 20,000 reads per cell.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
NextSeq 550 |
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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).
Reads from bc-Smart-seq2 single cells were demultiplexed based on cell barcodes using UMI-tools (version 0.5.3)
Reads without cell barcode or UMIs were removed and remaining raw reads were aligned to the human genome using HiSat2 (v.2.1.0) in single-end mode.
Primary counts were quantified with the function featureCounts (Subread version 1.6.0) using the flag –primary and -R BAM to save the BAM file.
PCR duplicates were removed and unique molecules were counted per gene and per cell using the function UMI-tools count
Genome_build: Homo.sapiens.GrCh38.91
Supplementary_files_format_and_content: A tab-delimited text file per sample containing gene counts of 1 pool (~84 single cells). Pools were combined into 20 superpools and used for bc-Smart-Seq2 single cell RNA sequencing (193 pools, n = 16 samples).
Reads from 10x Genomics Chromium single cells were demultiplexed and aligned to the GRCh38 genome with Ensembl transcriptome annotation with Cell Ranger using default parameters.
The demultiplexed fastq files were used as input for the 10x Genomics pipeline Cell ranger.
Barcode filtering was performed with the R package DropletUtils with a threshold of > 100 UMIs per barcode.
Genome_build: Homo.sapiens.GrCh38
Supplementary_files_format_and_content: A tab-separated barcodes text file, a tab-separated gene text file and a market exchange format (MEX) sparse count matrix per sample.
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 |
Mar 09, 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 |
SAMN14337380 |
| SRA |
SRX7878608 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4403148_2018-064_CTR_14-3_S1_completeCounts.txt.gz |
175.1 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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