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| Status |
Public on May 26, 2023 |
| Title |
EasySci-RNA, mouse brain |
| Sample type |
SRA |
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| Source name |
Mouse brain
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| Organism |
Mus musculus |
| Characteristics |
tissue: brain
strain: C57BL/6 background
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| Growth protocol |
The C57BL/6 WT mice and two AD models mutant mice were obtained from The Jackson Laboratory. All animal procedures were in accordance with institutional, state, and government regulations and approved under the IACUC protocol 21049.
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
EasySci-RNA: Extracted mouse brains were snap-frozen in liquid nitrogen and stored at -80°C. 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-ATAC: Mouse brain samples were snap-frozen in liquid nitrogen and stored at -80°C. For nuclei extraction, thawed brain samples were minced in PBS using a blade, re-frozen and stored at -80°C, and processed in multiple batches. On the day of nuclei extraction, minced tissues were resuspended in 1 mL EZ-sci-ATAC Nuclei Buffer (EZ-sci-ATAC NB) [10 mM Tris-HCl pH 7.5 (VWR, 97062-936), 10 mM NaCl (VWR, 97062-858), 3 mM MgCl2 (VWR, 97062-848), 0.1% Tween-20 (Sigma, P9416-100ML), 1x cOmplete™, EDTA-free Protease Inhibitor Cocktail (Sigma, 11873580001)], centrifuged at 200g for 5 minutes, resuspended in EZ-sci-ATAC NB supplemented with 0.1% IGEPAL® CA-630 (VWR, IC0219859650), incubated on ice for 5 minutes, and homogenized through 40 µm cell strainers (Fisher, 22363547) with the rubber tips of syringes inside a 6-cm dish (Genesee, 25-260). The isolated nuclei were centrifuged for 5 minutes at 500g, washed with 1 mL EZ-sci-ATAC NB, and re-suspended in 1 mL EZ-sci-ATAC NB. Nuclei concentration was determined by 4',6-diamidino-2-phenylindole staining (DAPI, Invitrogen D1306) and adjusted to ~10,000 nuclei/µL. Extracted nuclei were slow-frozen in 10% final concentration of DMSO, placed in FreezeCell containers (Genesee, 27-802) and stored at -80°C.
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). EasySci-ATAC: On the day of the library preparation, nuclei were thawed in a 37°C water bath and placed on ice immediately. Nuclei were washed once with EZ-sci-ATAC NB, filtered through pluriStrainer Mini 40 µm filters (Pluriselect, 43-10040-70), then concentrations were adjusted to 1000 nuclei/µL. Nuclei were mixed 1:1 ratio with 2X TD buffer [20 mM Tris-HCl pH 7.5, 20 mM MgCl2, 20% Dimethylformamide (Fisher, AC327175000)] and dispensed 10 µL (5000 nuclei) into each well of four 96-well plates. 1 µL barcoded Tn5 was loaded into each well. Tagmentation reaction was performed at 55°C for 10 minutes with gentle shaking at 300 rpm and stopped by adding 11 µL of 2X Stop buffer [40 mM EDTA (VWR, 97062-656), 1 mM Spermidine (Sigma, S0266-1G)] to each well. Samples were pooled and washed twice by EZ-sci-ATAC NB, then resuspended in 2.2 mL EZ-sci-ATAC NB. 5 µL tagmented sample (~4400 nuclei) was distributed into each well of four 96-well plates. 2 µL indexed EZ-sci P5 ligation adapters and 3 µL ligation mix [1 µL nuclease-free water, 1 µL 10X T4 DNA ligase buffer, 1 µL T4 DNA ligase (NEB, M0202L)] were added to each well. Ligation was performed at room temperature for 30 minutes with medium-speed rocking (350g) and stopped by adding 2 µL of 18 mM EDTA to each well. After that, nuclei were pooled, washed twice by EZ-sci-ATAC NB, resuspended in 300 µL EZ-sci-ATAC NB and filtered through a pluriStrainer Mini 20 µm filter (Pluriselect, 43-10020-70). The filter was washed three times with 100 µL EZ-sci-ATAC NB for maximum recovery rate. Then, nuclei were diluted to 2000 nuclei/µL and 5 µL (10000 nuclei) was distributed into 8-well PCR strips. Proteinase K treatment was performed by mixing each well with 0.25 μL 18.9 mg/mL proteinase K (Sigma, 3115828001), 0.25 µL 1% SDS and 0.5 µL EB buffer, and plates were incubated at 65°C for 16 hours. Then, 2 μL 10% Tween-20 was added to each well 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]GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-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, 11 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 by column purification using Zymo DNA Clean & Concentrator kit (Zymoresearch, D4014) followed by gel extraction using Zymoclean Gel DNA Recovery Kit (Zymoresearch, D4007) to remove adapter dimers. Library concentrations were determined by Qubit and the libraries were visualized by electrophoresis on a 2% E-Gel™ EX Agarose Gels (Invitrogen G402022).
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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 overalpped 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. EasySci-ATAC data processing: Base calls were converted to fastq format and demultiplexed using Illumina’s bcl2fastq/v2.19.0.316 tolerating one mismatched base in barcodes (edit distance (ED) < 2). Downstream sequence processing were similar to sci-ATAC-seq69. Indexed Tn5 barcodes and ligation barcodes were extracted, corrected to its nearest barcode (edit distance (ED) < 2) and reads with uncorrected barcodes (ED >= 2) were removed. Tn5 adaptors were removed from 5’-end and clipped from 3’-end using trim_galore/0.4.1 60. Trimmed reads were mapped to the mouse genome (mm39) using STAR/v2.5.2b 61 with default settings. Aligned reads were filtered using samtools/v1.4.1 70 to retain reads mapped in proper pairs with quality score MAPQ > 30 and to keep only the primary aligment. Duplicates were removed by picard MarkDuplicates/v2.25.271 per PCR sample. Deduplicated bam files were converted to bedpe format using bedtools/v2.30.072, which were further converted to offset-adjusted (+4 bp for plus strand and -5 bp for minus) fragment files (.bed). Deduplicated reads were further split into constituent cellular indices by further demultiplexing reads using the Tn5 and ligation indexes. For each cell, we also created sparse matrices counting reads falling into promoter regions (±1 kb around TSS) for downstream analysis. To define peaks of accessibility, we used MACS2/v2.1.176. Nonduplicate ATAC-seq reads of cells from each main cell type were aggregated and peaks were called on each group separately with these parameters: --nomodel --extsize 200 --shift -100 -q 0.05. To correct for differences in read depth or the number of nuclei per cell type, we converted MACS2 peak scores (−log10(q-value)) to ‘score per million’77 and filtered peaks by choosing a score-per-million cut-off of 1.3. Peak summits were extended by 250bp on either side and then merged with bedtools/v2.30.0. Cells were determined to be accessible at a given peak if a read from a cell overlapped with the peak. The peak count matrix was generated by a custom python script with the HTseq package78.
Assembly: mouse reference genome (mm39)
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; a cell annotation csv file, a peak annotation csv file, a peak count sparse matrix file, and a gene annotation csv file and a gene activity sparse matrix for ATAC data.
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| Submission date |
Sep 02, 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 |
GPL24247 |
| Series (1) |
| GSE212606 |
A global view of aging and Alzheimer’s pathogenesis-associated cell population dynamics in mammalian brain |
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| Relations |
| BioSample |
SAMN30654736 |
| SRA |
SRX17414909 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6538356_RNA_cell_annotation.csv.gz |
78.2 Mb |
(ftp)(http) |
CSV |
| GSM6538356_RNA_exon_annotation.csv.gz |
3.5 Mb |
(ftp)(http) |
CSV |
| GSM6538356_RNA_exon_count.txt.gz |
2.9 Gb |
(ftp)(http) |
TXT |
| GSM6538356_RNA_gene_annotation.csv.gz |
480.9 Kb |
(ftp)(http) |
CSV |
| GSM6538356_RNA_gene_count.txt.gz |
3.8 Gb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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