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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Jul 23, 2020 |
| Title |
Normal-1, Normal-2, T-M1Pool, T-M1T1, T-M1T2, T-M1T3, T-M1T4, and M-M1N1 |
| Sample type |
SRA |
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| Source name |
Whole Lung, Plucked Tumor, Metastasis
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| Organism |
Mus musculus |
| Characteristics |
strain: mixed; C57/BL/6J; 129/SV
tumor stage: normal, late-stage tumor, and metastasis
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| Treatment protocol |
Mice with tumors were induced at age 6-10 weeks. Mice were provided Adeno-SPC-Cre virus intratracheally resulting in recombination of Kras G12D, p53, and tdTomato in alveolar type II cells.
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| Growth protocol |
3T3 cells were grown in DMEM + 10% FBS + 1% pen/strep; GM12878 cells were grown in RPMI + 10% FBS + 1% pen/strep
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Lungs were dissociated with fine scissors and then proteolytic digestion was performed using the Lung Dissociation kit (Miltenyi Biotech) following the manufacturer’s instructions. Dissociated cells were then incubated at 37 degrees C for 20 minutes with rotation, then filtered using a 100-μm strainer. Red blood cells were lysed using ACK buffer (Thermo Scientific) and stained with APC-conjugated CD45 (BD, 559864), CD11b (eBioscience 17-0112-82), CD31 (Biolegend, 102510), Ter119 (BD, 557909), and DAPI (Sigma-Aldrich). Fluorescence-activated cell sorting (FACS) of immuno-stained primary cells was performed using a FACSAria sorter (BD) to isolate tdTomato+; DAPI-; APC- tumor cells for sciATAC-seq. Cells were then processed for sciATAC-Seq profiling following 0.1% fixation.
The PCR reaction was carried out at the following conditions: 72 °C for 5 minutes (extension), 98 °C for 5 min, and then thermocycling at 98 °C for 10 s, 70 °C for 30 s and 72 °C for 1 min. After thermocycling for 5 cycles, we took 5 µl sample from a few randomly selected wells and added 10 µl of PCR cocktail with 0.6x SYBRgreen. The 15 µl reactions were amplified to saturation to determine the number of cycles required for the remaining samples on the plate. Libraries were amplified for 13-14 cycles in total. Libraries were pooled and purified using Qiagen MinElute PCR purification column. The libraries were quantified using KAPA library quantification kit. Libraries were sequenced on the Next-seq platform (Illumina) using a 150-cycle kit (Read 1: 47 cycles, Index 1: 36 cycles, Index 2: 36 cycles, Read 2: 47 cycles).
sciATAC-seq
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| Library strategy |
ATAC-seq |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
sciATAC normal lung, plucked tumor, and metastasis
scATACseq_colData_new.txt
scATACseq_mouse_lung_counts.txt
scATACseq_mouse_lung_peaks.bed
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| Data processing |
Supplementary_files_format_and_content: Excel spreadsheet containing cell and sample-specific barcodes to demultiplex pooled sequence read files in .txt format. Columns include cell number, tissue source,lLouvain cluster, index1, and index2.
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| Submission date |
Jul 24, 2019 |
| Last update date |
Jul 23, 2020 |
| Contact name |
Lindsay M LaFave |
| E-mail(s) |
lmlafave@mit.edu
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| Organization name |
MIT
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| Department |
Biology
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| Lab |
Dr. Tyler Jacks
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| Street address |
77 Massachusetts Avenue; 76-453
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| City |
Cambridge |
| State/province |
MA |
| ZIP/Postal code |
02139 |
| Country |
USA |
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| Platform ID |
GPL19057 |
| Series (2) |
| GSE134812 |
Single-cell epigenomics identifies a premetastatic regulatory program in lung adenocarcinoma [sciATAC-seq] |
| GSE145194 |
Single-cell epigenomics identifies a premetastatic regulatory program in lung adenocarcinoma |
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| Relations |
| BioSample |
SAMN12358333 |
| SRA |
SRX6585778 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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