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| Status |
Public on Feb 09, 2023 |
| Title |
snRNA-seq of LAND-treated U87 and 3T3 nuclei with 10x Multiome (DEFND-seq RNA library) |
| Sample type |
SRA |
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| Source name |
U87 human glioma cell line / 3T3 mouse fibroblast cell line
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| Organisms |
Homo sapiens; Mus musculus |
| Characteristics |
cell line: U87 human glioma cell line / 3T3 mouse fibroblast cell line
cell type: human glioma cells / mouse fibroblasts
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| Growth protocol |
3T3 cells (ATCC #CRL-1658) and U87 MG cells (ATCC #HTB-14) were cultured in Dulbecco's Modified Eagle's Medium (ATCC #30-2002) supplemented with 10% feline bovine serum (FBS, ThermoFisher # A3160401). Eagle’s Minimum Essential Medium (ATCC #30-2003) was used to culture BJ cells (ATCC CRL-2522). TrypLE Express Enzyme (ThermoFisher #12605010) was used for dissociating cells during cell passages .
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
snATAC-seq: Nuclei were prepared from cryopreserved BJ fibroblasts. Frozen cells were thawed and added to a Dounce homogenizer containing 1 mL of nuclei isolation buffer (NIB: 10 mM TrisHCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% Igepal (Millipore Sigma #18896), and 1 x protease inhibitors (Millipore Sigma #118735800001)). The sample was gently homogenized on ice with 5 strokes of the loose pestle and 5 strokes of the tight pestle. The cells were transferred to a conical and 5 mL of PBS (Gibco #10010-031) was added. The sample was spun down at 500 g for 5 minutes, washed with 10 mL of PBS and resuspended in 100 µL of 10x Genomics Nuclei Buffer (10x Genomics, #2000207). xSDS snDNA-seq: Nuclei that were cross-linked and then treated with sodium dodecyl sulfate (SDS) to remove nucleosomes (xSDS) were prepared following Adey et al. Briefly, BJ fibroblasts were washed with 10 mL of PBS and covered in 3 mL of TrypLE (ThermoFisher #12605028) for 10 minutes at 37 °C. Then 9 mL of media was added, quenching the dissociation, and cells were spun down. The cells were resuspended in 10 mL of media with 406 µL of 37% formaldehyde (Millipore Sigma #252549) and gently rotated at room temperature for 10 minutes. 2.5 M glycine (800 µL) was then added and the suspension was incubated on ice for 5 minutes. Cells were centrifuged at 500 g for 8 minutes and washed with 10 mL of PBS. 5 mL of NIB was added to the pellet and the suspension was incubated for 20 minutes at 4 °C under rotation. Cells were then spun down at 500 g for 5 minutes and washed with 900 µL of NEBuffer 2.1 (New England Biolabs #B7202). The cells were pelleted (500 g for 5 minutes) and resuspended in NEBuffer 2.1 with 12 µL of 20% SDS. The solution was placed in a gentleMACS and vigorously shaken at 42 °C for 30 minutes. 200 µL of 10 % Triton-X (Millipore Sigma #T8787) was added and the cells were again shaken at 42 °C for 30 minutes. LAND snDNA-seq/DEFND-seq: The LAND protocol for adherent cells using 10x reagents was adapted from the LAND protocol. BJ fibroblasts were first washed with 10 mL of PBS and dissociated with 3 mL of TrypLE for 10 minutes at 37 C. Media (9 mL) was used to quench the dissociation reaction and collect the cells. The cells were spun down (300 g for 5 minutes) and 200 µL of DEFND buffer (175 µL NIB, 10 µL 1 mg/mL protease inhibitor (Millipore Sigma #11429868001), 25 µL 100 mM lithium diiodosalicylate (Millipore Sigma #653-14-5)), was added and incubated on ice for 5 minutes. Immediately following this incubation, 10 mL of nuclei isolation buffer was added and the nuclei were centrifuged at 4 °C for 5 minutes at 500 g. The supernatant was removed and 100 µL of 10x Genomics Nuclei Buffer. A fraction of nuclei was stained with SYBR green and counted on a Countess with a GFP filter set. GBM Cryopreserved Tissue DEFND-seq: A cryopreserved IDH wildtype Grade IV GBM sample was obtained from excess material collected for clinical purposes from de-identified brain tumor specimens. Nuclei were prepared from this anonymous sample according to XXXXX . Frozen tissue was placed in a Dounce homogenizer containing 1 mL of homogenization buffer (HB: 1 µM DTT, 250 mM sucrose, 25 mM KCl, 5 mM MgCl2, 10 mM tris, 1x protease inhibitor, 0.4 U/µL RNAse, 0.2 U/µL SUPERase In) and subjected to 15 strokes using the loose piston and 15 strokes using the tight piston. The homogenate was divided into 5 volumes and each was strained twice. The filtered homogenate was combined into a tube and centrifuged at 1000 g for 10 minutes at 4 °C. The pellet was resuspended in 1000 µL of HB and strained with 4 strainers (250 µL of homogenate for each strainer). The strained homogenate was again combined into a tube and centrifuged at 1000 g for 10 minutes at 4 °C. The pellet was resuspended in 250 µL of HB to which 250 µL of 50% iodixanol dilution media (IDM: 250 mM sucrose, 150 mM KCl, 30 mM MgCl2, 60 mM tris) was added resulting in a 25% IDM/nuclei suspension. This suspension was carefully layered on top of a tube containing 50% IDM at the bottom and 29% IDM at the surface, and centrifuged at 18,000 g for 23 minutes at 4 C. The nuclei formed a white pellet or sheet in the middle of the tube between the layers. All solution around this pellet was removed – first from the top and then from the bottom, and the pellet was resuspended in 1 mL of chilled PBS and centrifuged (500 g for 5 minutes at 4 °C). The supernatant was removed and the pellet was then treated with DEFND buffer for 5 minutes on ice. Immediately following this incubation, 5 mL of NIB with RNAse inhibitors was added at the nuclei were centrifuged at 500 g for 5 minutes at 4 °C.
snATAC-seq: Single-cell ATAC libraries were prepared with these nuclei using the 10x Genomics Single Cell ATAC kit (10x Genomics #1000176, #1000162, and #1000212) according to the manufacturer’s protocol. xSDS snDNA-seq: The cells were pelleted (500 g for 5 minutes) and resuspended in 100 µL of 10x Genomics Nuclei Buffer, counted, and tagmented following the Chromium Single Cell ATAC protocol. Tagmented nuclei were loaded onto a 10x Chromoium controller and libraries were prepared according to the Chromimum Single Cell ATAC protocol (10x Genomics #1000176, #1000162, and #1000212). LAND snDNA-seq: Nuclei were then tagmented and prepared for single-cell sequencing with the Chromium Single Cell ATAC kit (10x Genomics #1000176, #1000162, and #1000212) and protocol. DEFND-seq: Nuclei were then tagmented and prepared for single-cell sequencing with the Chromium Single Cell Multiome kit (10x Genomics #1000285, #1000230, #1000215).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic single cell |
| Library selection |
cDNA |
| Instrument model |
NextSeq 550 |
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| Description |
3T3-U87-Mix-RNA
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| Data processing |
snRNA-seq data were processed as described previously [Yuan and Sims, Scientific Reports, 2016]. The code is available at (https://github.com/simslab/DropSeqPipeline8). Briefly, we aligned the transcriptomic reads (read 2) to the human or mouse genome and transcriptome annotation using STAR v2.7.0d [Dobin et al, Bioinformatics, 2013]. For human data sets other than the mixed species experiment, we used GRCh38/Gencode v24. For the mixed species data set, we used a concatenated genome and annotation comprised of GRCh38/Gencode v32 and GRCm38/Gencode vM23. Because we are analyzing nuclei that contain a significant amount of unspliced mRNA, we identified reads that uniquely and strand-specifically aligned to the entire gene body of each gene (including exons, introns, intron-exon junctions, and exon-exon junctions), assigning an address to each aligned read containing the gene name, cell-identifying barcode sequence, and unique molecular identifier (UMI) extracted from read 1. Next, we demultiplexed the resulting table, correcting sequencing errors in the barcodes as described in Yuan and Sims [Yuan and Sims, Scientific Reports, 2016] to produce a count matrix for each sample.
snDNA-seq and snATAC-seq data were processed using a custom pipeline available here (https://github.com/simslab/dna10x). The pipeline attempts to mimic some of the basic data processing procedures that are implemented in the 10x Genomics Cell Ranger software packages for analysis of snATAC-seq and Multiome data, including the formatting of output files. We generated sample-demultiplexed fastq files using the mkfastq command in cellranger-atac v2.0.0 (10x Genomics). We aligned paired-end reads 1 and 2 to the human or mouse genome using bwa mem v0.7.17-r1188 [Li, arXiv, 2013] after removing a standard adapter sequence (CTGTCTCTTATACACATCT) from each read using cutadapt v2.8 [Martin, EMBnet.journal, 17, 1, 2011]. We then extracted all reads with an alignment score that was >90% of the read length and insert size <1 kb. We also implemented an error correction procedure for the cell-identifying barcodes from read 3. Briefly, for cell-identifying barcodes that do not appear in the standard list provided by 10x Genomics, we first determine the frequency falt of each barcode in the standard list in the data set. For each observed barcode sequence that has a Hamming distance of one from a sequence on the standard list, we estimate the posterior probability palt that the barcode deviates from the standard list due to sequencing error using the quality scores for the putatively errant base provided by Illumina or Element palt = falt10-q/10 where q is the quality score. If palt>0.975 for any alternative barcodes, then we replace the errant barcode with the alternative barcode from the standard list with the highest value of palt. Finally, we establish read addresses for each alignment that passes the filter described above comprised of a cell-identifying barcode, chromosome, a fragment start position, a fragment end position and collapse identical fragments. Finally, we sort the fragments by alignment position on the genome and output a table in the format of the “fragments.tsv” file that is typically produced by Cell Ranger.
Assembly: human-only libraries: GRCh38/Gencode v24; human/mouse mixture: GRCm38/Gencode vM23/GRCh38/Gencode v32
Supplementary files format and content: PTO016PTO021.fragments.tsv.bz2 - tab-delimited file tabulating the alignment position (chromosome, start, end), cell-identifying barcode, and read count for each unique fragment for genomic data (scATAC-seq, scDNA-seq, DEFND-seq)
Supplementary files format and content: PTO017_xSDS.fragments.tsv.bz2 - tab-delimited file tabulating the alignment position (chromosome, start, end), cell-identifying barcode, and read count for each unique fragment for genomic data (scATAC-seq, scDNA-seq, DEFND-seq)
Supplementary files format and content: PTO020.fragments.tsv.bz2 - tab-delimited file tabulating the alignment position (chromosome, start, end), cell-identifying barcode, and read count for each unique fragment for genomic data (scATAC-seq, scDNA-seq, DEFND-seq)
Supplementary files format and content: PTO018.matrix.txt.gz - tab-delimited file containing the scRNA-seq count matrix where each row is a gene (labeled by a gene ID and gene symbol) and each column is a cell (with a header containing the cell-identifying barcode and cluster number)
Supplementary files format and content: PTO025.fragments.tsv.bz2 - tab-delimited file tabulating the alignment position (chromosome, start, end), cell-identifying barcode, and read count for each unique fragment for genomic data (scATAC-seq, scDNA-seq, DEFND-seq)
Supplementary files format and content: 3T3-U87-Mix.matrix.txt.gz - tab-delimited file containing the scRNA-seq count matrix where each row is a gene (labeled by a gene ID and gene symbol) and each column is a cell (with a header containing the cell-identifying barcode and cluster number)
Supplementary files format and content: PTO034.fragments.tsv.bz2 - tab-delimited file tabulating the alignment position (chromosome, start, end), cell-identifying barcode, and read count for each unique fragment for genomic data (scATAC-seq, scDNA-seq, DEFND-seq)
Supplementary files format and content: TB5471.matrix.txt.gz - tab-delimited file containing the scRNA-seq count matrix where each row is a gene (labeled by a gene ID and gene symbol) and each column is a cell (with a header containing the cell-identifying barcode and cluster number)
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| Submission date |
Jan 31, 2023 |
| Last update date |
Oct 08, 2023 |
| Contact name |
Peter A Sims |
| E-mail(s) |
pas2182@columbia.edu
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| Organization name |
Columbia University
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| Street address |
3960 Broadway, Lasker 203AC
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| City |
New York |
| State/province |
NY |
| ZIP/Postal code |
10032 |
| Country |
USA |
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| Platform ID |
GPL24625 |
| Series (1) |
| GSE224149 |
Scalable co-sequencing of RNA and DNA from individual nuclei |
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| Relations |
| BioSample |
SAMN32979124 |
| SRA |
SRX19235310 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7016417_3T3-U87-Mix.matrix.txt.gz |
25.1 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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