|
| Status |
Public on Jan 01, 2023 |
| Title |
b3_1223um |
| Sample type |
SRA |
| |
|
| Source name |
Spatial transcriptome of the layer 1223 micrometers away from biofilm edge in biofilm 3
|
| Organism |
Escherichia coli BW25113 |
| Characteristics |
growth mode: biofilm
|
| Growth protocol |
For biofilm growth, single colony was picked up from a stock LB agar plate and was grown aerobically with shaking at 220 rpm in LB medium overnight (typically 12 hours) as the seed culture. We sub-cultured 100 uL seed into 5 mL fresh LB broth (2% volume) in 50 mL centrifuge tube and grown for two hours at 37 ℃ with shaking at 220 rpm to reach exponential stage. Next, bacteria would be loaded into microfluidic chip to grow a semicircular biofilm. When the radius of biofilm reached around 1 mm, bacteria cells would be extracted from the chip for further steps. To prepare planktonic bacterial culture, single colony was picked up from a stock LB agar plate and was grown aerobically with shaking at 220 rpm in LB medium overnight (typically 12 hours) as the seed culture. A subculture was established by cultivating the seed culture into fresh 5 mL fresh LB broth (2% volume) in 50 mL centrifuge tube and grown for two hours with shaking at 220 rpm to reach exponential stage (OD600 ~ 0.6). Such subculture was next inoculated into 300 uL M63B1 broth in 48-well plate. The 48-well plate were loaded into a microplate reader system with 220 rpm shaking and the growth curve was monitored from absorbance at 600 nm every 30 min. When reaching desired OD600, 100 uL culture was taken to extract RNA.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Bacterial RNA was obtained utilizing the RNAClean XP beads (Beckman, A63987). Lysate was prepared using lysozyme and proteinase K
miniBac-seq, a customized bacterial RNA-seq library construction method, see description of our paper for details
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Description |
b3_1223um
E. coli k12 BW25113
|
| Data processing |
quality trimming step using cutadapt (v2.10) command with a quality threshold of 20
the pooled sample was demultiplexed according to the anchored barcode of the first seven nucleotides at the 5’-end from the first read
internal adaptor sequence embedding in some second reads with too short insert was also trimmed
a minimum length threshold of 20 bp for both the first and second reads was used to filter out those pair-end reads with too short insert
bowtie2 (v2.4.1) to align the second read to a reference genome (NC000913.3) (default setting)
Feature counting was carried out using R method summarizeOverlaps from GenomicAlignments package (v1.26.0) in a strand-specific manner, giving rise to a read count table for each gene.
Genome_build: NC000913.3
Supplementary_files_format_and_content: gene raw count data, tab-delimited matrix table
|
| |
|
| Submission date |
Feb 27, 2022 |
| Last update date |
Jan 01, 2023 |
| Contact name |
Tianmin Wang |
| E-mail(s) |
wangtm@shanghaitech.edu.cn
|
| Organization name |
ShanghaiTech University
|
| Department |
School of Life Science and Technology
|
| Lab |
Tianmin Wang Lab
|
| Street address |
Huaxia Middle Road, 393
|
| City |
Shanghai |
| ZIP/Postal code |
201210 |
| Country |
China |
| |
|
| Platform ID |
GPL27123 |
| Series (1) |
| GSE197541 |
RAINBOW-seq to capture the spatial transcriptome of bacterial biofilm |
|
| Relations |
| BioSample |
SAMN26287472 |
| SRA |
SRX14305910 |
