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Sample GSM6994841 Query DataSets for GSM6994841
Status Public on Feb 06, 2023
Title E coli and B subtilis barnyard experiment
Sample type SRA
 
Source name Bacterial cells in suspension
Organisms Escherichia coli; Bacillus subtilis
Characteristics cell type: Bacterial cells in suspension
strain: BS 168 and Ec_MG1655
age: Mid log phase
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.
 
Library strategy RNA-Seq
Library source transcriptomic single cell
Library selection cDNA
Instrument model NextSeq 2000
 
Description RNA hybridizing probes
10x Genomics
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
 
Submission date Jan 26, 2023
Last update date Feb 06, 2023
Contact name Adam Rosenthal
E-mail(s) adam.z.rosenthal@gmail.com
Phone 6263939102
Organization name UNC
Department Microbiology and Immunology
Lab Adam Rosenthal
Street address 125 Mason Farm Marsico Hall rm6201
City Chapel Hill
State/province NC
ZIP/Postal code 27599
Country USA
 
Platform ID GPL33060
Series (1)
GSE223752 probe based bacterial single-cell RNA sequencing predicts toxin regulation
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 SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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