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Status |
Public on Feb 06, 2023 |
Title |
E coli and B subtilis barnyard experiment |
Sample type |
SRA |
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Source name |
Bacterial cells in suspension
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Organisms |
Escherichia coli; Bacillus subtilis |
Characteristics |
cell type: Bacterial cells in suspension strain: BS 168 and Ec_MG1655 age: Mid log phase
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Extracted molecule |
total RNA |
Extraction protocol |
Freshly collected bacterial cells were fixed in 1% paraformaldehyde for 1 hour. Cells from 2 mL of cell culture were fixed using a 30-minute incubation in 1% paraformaldehyde (final concentration) at room temperature. Formaldehyde-fixed samples were washed with 0.02 % SSC (Saline Sodium Citrate - Invitrogen) by gentle centrifugation (6,000 x G) for 2.5 minutes. After the wash, cell pellets were resuspended in 1 mL MAAM (4:1 V:V dilution of methanol to glacial acetic acid). Samples were kept at -20oC for up to 2 days before further processing. For in-situ probe hybridization, 150 µL of fixed sample was centrifuged and washed once in PBS to remove methanol and acetic acid. After the wash step, cells were incubated in 200 µL PBS with 350 u/µL of lysozyme solution (Epicentre ready-lyse) for 30 minutes at room temperature. Lysozyme concentration was optimized and other enzyme combinations were tested to achieve a protocol with maximal probe signal and minimal cell lysis (see Supplementary Figure S12) After 30 minutes cells were pelleted and washed with 500 µL PBS-tween (PBS with 0.1 % Tween 20). Cells were then re-suspended with 100 µL of probe binding buffer consisting of 5x SSC, 30% formamide, 9mM citric acid (pH 6.0). 0.1% Tween 20, 50 ug/ml heparin, and 10% low MW dextran sulfate42. Cell suspensions were placed in a 50oC shaker-incubator and allowed to pre-equilibrate for 1 hour. After 1 hour 50 µL of probes, (600 ng/µL) were added to each cell suspension, and samples were left to incubate overnight. Following the overnight incubation samples were washed 5 times in pre-warmed (50oC) probe-wash solution (5 x SSC, 30% formamide, 9 mM citric acid pH 6.0, 0.1% Tween 20, and 50 ug/mL heparin). Library was performed according to specifications of McNulty et al bacterial scRNAseq protocols (single cell 3’ v2 protocol, 10x Genomics). Briefly, Single cell partitioning, barcoding, and cDNA library generation was achieved using the 10X Genomics Chromium Controller with the Chromium Single Cell 3’ Reagents Kit (v2 chemistry) as described by 10X Genomics (https://support.10Xgenomics.com/single cell-gene-expression/index/doc/user-guide-chromium-single cell-3-reagent-kits-user-guide-v2-chemistry). The protocol was modified to achieve bacterial scRNA-seq. For GEM generation (10X microfluidic encapsulation), a master mix containing the following reagents (per rxn, not accounting for excess volume) was prepared: 33 µL of 4X ddPCR Multiplex Supermix (BioRad), 4 µL of custom primer (10 µM), 2.4 µL additive A (10X Genomics), 26.8 µL dH2O. All other reagents specified by 10X Genomics were omitted. Prepared cell samples were washed 3X in PBS and diluted to 1000 cells/µL before loading on the microfluidic chip (“Chip A Single Cell”) with a targeted cell recovery of 10,000 cells.
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Library strategy |
RNA-Seq |
Library source |
transcriptomic single cell |
Library selection |
cDNA |
Instrument model |
NextSeq 2000 |
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Description |
RNA hybridizing probes 10x Genomics
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Data processing |
The demultiplexing, barcoded processing, gene counting and aggregation were made using the Cell Ranger software V7.1 and using custom scripts available from the Sahand Hormoz lab (https://gitlab.com/hormozlab/bacteria_scrnaseq) Assembly: Probe-based genome builds for B subtilis, E coli and C perfringens are deposited alongside GTF files in the Hormoz lab github: https://gitlab.com/hormozlab/bacteria_scrnaseq Supplementary files format and content: H5 files
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Submission date |
Jan 26, 2023 |
Last update date |
Feb 06, 2023 |
Contact name |
Adam Rosenthal |
E-mail(s) |
adam.z.rosenthal@gmail.com
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Phone |
6263939102
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Organization name |
UNC
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Department |
Microbiology and Immunology
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Lab |
Adam Rosenthal
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Street address |
125 Mason Farm Marsico Hall rm6201
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City |
Chapel Hill |
State/province |
NC |
ZIP/Postal code |
27599 |
Country |
USA |
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Platform ID |
GPL33060 |
Series (1) |
GSE223752 |
probe based bacterial single-cell RNA sequencing predicts toxin regulation |
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Relations |
BioSample |
SAMN32925743 |
SRA |
SRX19186222 |
Supplementary file |
Size |
Download |
File type/resource |
GSM6994841_Barnyard_filtered_feature_bc_matrix.h5 |
10.4 Mb |
(ftp)(http) |
H5 |
SRA Run Selector |
Raw data are available in SRA |
Processed data provided as supplementary file |
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