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| Status |
Public on May 26, 2023 |
| Title |
EasySci-RNA, human brain |
| Sample type |
SRA |
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| Source name |
Human brain
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| Organism |
Homo sapiens |
| Characteristics |
tissue: brain
brain region: mixed sample (non-demultiplexed)
diagnosis: mixed sample (non-demultiplexed)
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| Growth protocol |
Twenty-four post-mortem human brain samples across two regions (hippocampus and superior and middle temporal gyrus) and twelve individuals, including six controls and six Alzheimer's disease patients ranging from 70 to 94 in age, were collected from the University of Kentucky Alzheimer's Disease Center Tissue Bank.
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
EasySci-RNA: Twenty-four post-mortem human brain samples across two regions (hippocampus and superior and middle temporal gyrus) and twelve individuals, including six controls and six Alzheimer's disease patients ranging from 70 to 94 in age, were collected from the University of Kentucky Alzheimer's Disease Center Tissue Bank. Each surveyed sample underwent rigorous quality control, including short PMI, and was subjected to EasySci-RNA profiling. For nuclei extraction, thawed samples were minced in 1 mL PBS (Genesee, 25-507) with 1% Diethyl Pyrocarbonate (DEPC) (VWR, 97062-652) using a blade on a 6 cm dish (Genesee, 25-260) on ice. After centrifuging for 5 minutes at 200g (4 °C), 1mL ice-cold EZ lysis buffer (Millipore Sigma, NUC101-1KT) +1% DEPC was added to the tissue (0.1g-0.5g) for nuclei extraction and kept on ice for 5 minutes. The nuclei suspension was homogenized through 40 µm cell strainers (VWR, 470236-276) with the rubber tips of syringes inside a 6-cm dish, centrifuged for 5 minutes at 500g, re-suspended in 500ul EZ lysis buffer + 0.1% SuperRnase Inhibitor (Thermo Fisher Scientific, AM2696). After repeated centrifugation for 5 minutes at 500g, the nuclei are fixed using 1mL ice-cold 0.1% Formaldehyde (Thermofisher, 28906) on ice for 10 minutes. The fixed nuclei are centrifuged for 3 minutes at 500g, resuspended in 500ul EZ buffer + 0.1% SuperRnase Inhibitor, centrifuged again for 5 minutes at 500g, and re-suspended in 500mL EZ buffer + 0.1% SuperRnase Inhibitor. After checking the nuclei morphology, the nuclei are re-suspended in 100uL nuclei suspension buffer (NSB) [Nuclei Buffer (10mM Tris-HCl, pH 7.5 (Thermo Fisher Scientific, 15567027); 10mM NaCl (Thermo Fisher Scientific, AM9759); 3mM MgCl2 (Thermo Fisher Scientific, AM9530G) in nuclease-free water (Ambion, AM 9937)) with 1% SuperRnase Inhibitor and 1% BSA (NEB, B9000S)] + 10% Dimethyl Sulfoxide (DMSO) (VWR, 97063-136) and slow frozen to -80C for storage.
EasySci-RNA: On the day of the library preparation, nuclei were thawed in a 37°C water bath and placed on ice immediately. After thawing, 500 μL NSB + Triton X-100 (Sigma Aldrich, 93443-100ML) is added, and the nuclei are sonicated for 12 seconds at low power. After filtering the nuclei through a 20 μm filter (PluriSelect 43-10020-70) the sample is pelletized for 5 minutes, 500 g at 4°C and resuspended in 100 μL of NSB. After nuclei counting the concentration is set to 20,000 nuclei/μL. For the reverse transcription reaction nuclei in 4 μL of NSB, 0.5 μL of 10 mM dNTP (Thermo Fisher Scientific, R0192), 1 μL 50 μM short-dT primer (100 μM, 5′-/5Phos/ACGACGCTCTTCCGATCTNNNNNNNN[10bp barcode]TTTTTTTTTTTTTTTT-3′, where “N” is any base; IDT) and 1μL 50 μM randomN primer (100 μM, 5'-/5Phos/ACGACGCTCTTCCGATCTNNNNNNNN[10bp barcode]NNNNNN-3', where "N" is any base; IDT) are distributed on every well of a 96-well plate and incubated at 55°C for 5 minutes. 3.5 μL of reverse transcription reaction mix [Maxima H Minus Reverse Transcriptase (105 μL) with Buffer (420 μL) (ThermoFisher, EP0753); 105 μL SuperRnase Inhibitor; 105 μL nuclease-free water] is added to each well and the following thermocycler program is used for the reaction: 4°C for 2 minutes, 10°C for 2 minutes, 20°C for 2 minutes, 30°C for 2 minutes, 40°C for 2 minutes, 50°C for 2 minutes, 55°C for 15 minutes. After the reverse transcription reaction, 10 μL NBB (Nuclear Buffer + 1% BSA + 0.1% Triton-X-100) is added to each well, the wells are pooled and moved into a 15 mL tube and centrifuged for 3 minutes, 1000 g at 4°C. Then the nuclei is resuspended in 1 mL NBB, moved into a 1.5 mL microcentrifuge tube, and centrifuged again for 3 minutes, 1000 g at 4°C. For the ligation reaction, the cells are resuspended in 950 μL NBB, and distributed into four PCR plates with 2.5 μL of the solution going into each well. Then 1 μL of the DNA ligation primer(100 μM, 5’-AATGATACGGCGACCACCGAGATCTACAC[10bp barcode]ACACTCTTTCCCTAC-3’, IDT) + adaptor (100 μM,5'-A*G*A*T*C*G*G*A*A*G*A*G*C*G*T*C*G*T*G*T*A*G*G*G*A*A*A*G*A*G*T*G*T*/3ddC/, IDT) complex (3.125 μM) is added with 1.5 μL of the ligation reaction mix [210 μL 10x T4 Ligation Buffer with 210 μL T4 DNA Ligase (NEB, M0202L); 21 μL SuperRnase Inhibitor; 189 μL nuclease free water] and the mixture is incubated for 30 minutes at room temperature with gentle shaking (300 rpm with Thermomixer, 50 rpm on Fisherbrand Nutating Mixer). After 30 minutes, 1 μL EDTA (18 mM) (VWR, 97062-656) is added to each well, and the wells are pooled into a 15 mL tube. After centrifuging the tube for 3 minutes, 1000 g at 4°C, the nuclei are resuspended in 1 mL NBB, centrifuged again for 3 minutes, 1000 g at 4°C, resuspended in 500 μL NBB and filtered through a 40 μM filter and centrifuged again for 3 minutes, 1000 g at 44°C and resuspended in 500 μL NBB for nuclei counting. After counting, 10,000 nuclei per well is distributed in 4 μL / well volume on a 96 well plate. The second-strand synthesis is performed by adding 1 μL enzyme-buffer mix [⅔ μL Second-Strand Synthesis buffer + ⅓ μL Second-Strand Synthesis Enzyme Mix (NEB, E7550S)] and incubating at 16°C for one hour, followed by 0.8X AMPure beads (Beckman Coulter, A63882) purification. For the tagmentation step, 6.6 μL of the tagmentase-buffer mix (Nextera N7 adaptor loaded Tn5 (provided by Illumina) or custom Tn5) is added and the solution is incubated at 55°C for 5 minutes. Next, 0.4 µL 1% SDS (ThermoFisher AM9820) and 0.4 µL BSA is added and incubated at 55°C for 15 minutes to stop the tagmentation reaction. Then, 2 μL 10% Tween-20 (Millipore Sigma, P9416-100ML) was added to quench the SDS. Following on, 1 μL of 10 μM universal P5 primer (5′-AATGATACGGCGACCACCGAGATCTACAC-3′, IDT), 1 μL of 10 μM indexed P7 primer (5′-CAAGCAGAAGACGGCATACGAGAT[i7]GTCTCGTGGGCTCGG-3′, IDT) and 10 μL NEBNext High-Fidelity 2X PCR Master Mix (NEB, M0541L) were added into each well. Amplification was carried out using the following program: 72°C for 5 minutes, 98°C for 30 seconds, 12-15 cycles of (98°C for 10 seconds, 66°C for 30 seconds, 72°C for 30 seconds) and a final 72°C for 5 minutes. Final PCR products were pooled and purified with 0.8X AMPure beads and the second round of 0.9X AMPure beads to remove primer dimers. Library concentrations were determined by Qubit and the libraries were visualized by electrophoresis on a 2% E-Gel™ EX Agarose Gels (ThermoFisher, G402002).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic single cell |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
EasySci-RNA library
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| Data processing |
EasySci-RNA data processing: A custom computational pipeline was developed to process the raw fastq files from the EasySci libraries. Similar to our previous studies10,11, the barcodes of each read pair were extracted. Both adaptor and barcode sequences were trimmed from the reads. Second, an extra trimming step is implemented using Trim Galore 60 with default settings to remove the poly(A) sequences and the low-quality base calls from the cDNA. Afterward, the paired-end sequences were aligned to the genome with the STAR aligner 61, and the PCR duplicates removed based on the UMI sequence and the alignment location. Finally, the reads are split into SAM files per cell, and the gene expression is counted using a custom script. At this level, the reads from the same cell originating from the short dT and the random hexamer RT primers were counted as independent cells. During the gene counting step, we assigned reads to genes if the aligned coordinates overlapped with the gene locations on the genome. If a read was ambiguous between genes and derived from the short dT RT primer, we assigned the read to the gene with the closest 3’ end; otherwise, the reads were labeled as ambiguous and not counted. If no gene was found during this step, we then searched for candidate genes 1000 bp upstream of the read or genes on the opposite strand. Reads without any overlapped genes were discarded. We used a similar strategy to generate an exon count matrix across cells. Specifically, we counted the number of expressed exons based on the number of reads overlapping each exon. If one read overlapped with multiple exons, this read was split between the exons. Read overlapped with multiple genes were discarded, except if we can determine the exact gene based on the other paired-end read. For reads without overlapped genes, we checked if there are any overlapped exons on the opposite strand. Reads without any overlapped exons were discarded.
Assembly: human reference genome (hg38)
Supplementary files format and content: Processed data files include a cell annotation csv file, a gene annotation csv file, and a gene count sparse matrix file for RNA data.
Supplementary files format and content: cell_annotation.csv: Cell annotation file: sample is cell id of each single cell with the ligation and reverse transcription barcode attached; UMAP_1 and UMAP_2 are cell coordinates after UMAP dimension reduction; Cell_type contains the cell type annotation.
Supplementary files format and content: gene_annotation.csv: Gene annotation file including gene id, gene type and gene short name.
Supplementary files format and content: gene_count.txt: A sparse matrix file: each row corresponds to gene id (with gene information in the gene_annotation.csv); each column corresponds to each cell (with cell source information in the cell_annotation.csv); each value in the matrix corresponds to UMI count.
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| Submission date |
Oct 18, 2022 |
| Last update date |
May 27, 2023 |
| Contact name |
Junyue Cao |
| E-mail(s) |
jcao@rockefeller.edu
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| Organization name |
The Rockefeller University
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| Lab |
Cao lab
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| Street address |
1230 York Avenue
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| City |
New York |
| State/province |
NY |
| ZIP/Postal code |
10065 |
| Country |
USA |
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| Platform ID |
GPL24676 |
| Series (1) |
| GSE212606 |
A global view of aging and Alzheimer’s pathogenesis-associated cell population dynamics in mammalian brain |
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| Supplementary file |
Size |
Download |
File type/resource |
| GSM6657986_cell_annotation.csv.gz |
3.6 Mb |
(ftp)(http) |
CSV |
| GSM6657986_gene_annotation.csv.gz |
564.0 Kb |
(ftp)(http) |
CSV |
| GSM6657986_gene_count.txt.gz |
387.7 Mb |
(ftp)(http) |
TXT |
| Raw data not provided for this record |
| Processed data provided as supplementary file |
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