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Sample GSM8128999

Query DataSets for GSM8128999

Status

Public on Sep 18, 2024

Title

Mn_US3

Sample type

SRA

Source name

ATCC 43300

Organism

Staphylococcus aureus

Characteristics

cell line: ATCC 43300
genotype: WT
treatment: Treated by MnPM+US

Treatment protocol

After adding 100 μg/mL nanomaterials into the bacterial suspension of each group, the bacteria were incubated for 12 hours. Bacterial precipitation was obtained by centrifugation.

Growth protocol

The frozen Staphylococcus aureus (ATCC 43300) strain was defrosted at room temperature and cultured in trypsin soybean broth medium (TSBG) containing 0.25% glucose through inoculation ring dipped in bacterial solution. After overnight in a shaking bed at 37℃, a logarithmic growth phase bacterial solution was formed. The gradient dilution contained 109 CFU/mL bacterial suspension, and the dilution was 100 times to produce 107 CFU/mL working bacterial solution. The sterilized aseptic titanium sheet was placed into the 24-well plate, 800μL of working bacterial solution was taken and added into the 24-well plate, and then placed in a 37℃ temperature box. Carefully remove the orifice plate and carefully update the TSBG medium every 24 hours. After 48 hours of culture, the supernatant medium was sucked out, and the titanium sheet attached to the biofilm was found in the 24-well plate.

Extracted molecule

total RNA

Extraction protocol

A. Add 600 μL Lysis/Binding Buffer to the cell or tissue lysate. Then add 30 μL miRNA Homogenate Additive to homogenate, and mix well by vortexing or inverting the tube several times. Leave the mixture on ice for 10 min. B. Add a volume of Acid-Phenol: Chloroform that is equal to the lysate volume before addition of the miRNA Homogenate Additive. Centrifuge for 5 min at maximum speed (10,000 x g) to separate the aqueous and organic phases. Carefully remove the aqueous (upper) phase without disturbing the lower phase, and transfer it to a fresh tube. C. Add 1.25 volumes of room temperature 100% ethanol to the aqueous phase. D. Pipet the lysate/ethanol mixture (from the previous step) onto the Filter Cartridge (Note: Up to 700 μL can be applied to a Filter Cartridge at a time.). Centrifuge for 30 sec at 13,000 rpm to pass the mixture through the filter. Discard the flow-through. E. Apply 350 μL miRNA Wash Solution 1 to the Filter Cartridge and centrifuge for 30 sec at 13,000 rpm. Discard the flow-through from the Collection Tube, and replace the Filter Cartridge into the same Collection Tube. F. The 10 μL DNase I and 70 μL Buffer RDD QIAGEN (#79254) were mixed. Then the mixture was added to the Filter Cartridge. Leave it at the room temperature for 15 min. G. Apply 350 μL miRNA Wash Solution 1 to the Filter Cartridge and centrifuge for 30 sec at 13,000 rpm. Discard the flow-through from the Collection Tube, and replace the Filter Cartridge into the same Collection Tube. H. Apply 500 μL Wash Solution 2/3 and centrifuge for 30 sec at 13,000 rpm. Draw it through the Filter Cartridge as in the previous step. Repeat with a second 500 μL aliquot of Wash Solution 2/3. I. Spin the assembly for 1 min to remove residual fluid from the filter. Transfer the Filter Cartridge into a fresh Collection Tube. Apply 100 μL of pre-heated (95°C) Elution Solution to the center of the filter. Leave it at the room temperature for 2 min. Spin for 20–30 sec at maximum speed to recover the RNA. Collect the eluate (which contains the RNA) and store it at -70°C.
a. Add 5 μL rRNA Binding Buffer and 5 μL rRNA Removal Mix-Gold to each well with 10 μL total RNA (1 μg). Gently pipette the entire volume up anddown 6 times to mix thoroughly. b. Incubate the PCR plate at 68°C for 5 min, and then place at room temperature for 1 min. c. Add 35 μL of rRNA Removal Beads to each well of the new PCR plate. Adjust the pipette to 45 μL, then with the tip of the pipette at the bottom of the well, pipette quickly up and down 20 times to mix thoroughly. Incubate at room temperature for 1 min. Place the PCR plate on the magnetic stand at room temperature for 1 min. Transfer all of the supernatant from each well to the corresponding well of the new PCR plate. d. Add 99 μL of RNAClean XP beads (If starting with degraded total RNA, add 193 μL of RNAClean XP beads to each well) to each well of the PCR plate. Gently pipette the entire volume up and down 10 times to mix thoroughly. Incubate the PCR plate at room temperature for 15 min. e. Place the PCR plate on the magnetic stand at room temperature for 5 min. Remove and discard all of the supernatant from each well of the PCR plate. f. Add 200 μL freshly prepared 70% EtOH to each well. Incubate the PCR plate at room temperature for 30 s, and then remove and discard all of the supernatant from each well. Let the PCR plate stand at room temperature for 15 min to dry. g. Add 11 μL Elution Buffer to each well of the PCR plate. Gently pipette the entire volume up and down 10 times to mix thoroughly. Incubate the PCR plate at room temperature for 2 min. Place the PCR plate on the magnetic stand at room temperature for 5 min. Transfer 8.5 μL supernatant from the PCR plate to the new 0.2 ml tube. h. Add 8.5 μL Elute, Prime, Fragment High Mix to each well of the plate. Gently pipette the entire volume up and down 6 times to mix thoroughly. i. Incubate at 94°C for 8 min, hold at 4°C, and then centrifuge shortlya. Add 8 μL First Strand Synthesis Act D Mix and SuperScript II Reverse Transcriptase in each well of the PCR plate. Gently pipette the entire volume up and down 6 times to mix thoroughly. b. The mixture was incubated in the following progress: 25°C for 10 min, 42°C for 15 min, 70°C for 15 min, hold at 4°C.
2.3 Synthesize Second Strand cDNA a. Add 5 μL End Repair Control (2 μL End Repair Control + 98 μL Resuspension Buffer) in each well of the PCR plate. b. Add 20 μL Second Strand Marking Master Mix. Gently pipette the entire volume up and down 6 times to mix thoroughly. c. Incubate at 16°C for 60 min. Remove the plate from the thermal cycler2.3.1 Purification a. Add 90 μL mixed AMPure XP beads to each well of the plate. Gently pipette the entire volume up and down 10 times to mix thoroughly. Incubate the plate at room temperature for 15 min. b. Place it on the magnetic stand at room temperature for 5 min. c. Remove and discard 135 μL supernatant from each well of the plate. d. With the plate on the magnetic stand, add 200 μL freshly prepared 80% EtOH to each well. Incubate the plate at room temperature for 30 s, and then remove and discard all of the supernatant from each well. e. Repeat step d one time. f. Let the plate stand at room temperature for 15 min to dry, and then remove the plate from the magnetic stand. g. Add 17.5 μL Resuspension Buffer to each well of the plate. Gently pipette the entire volume up and down 10 times to mix thoroughly. h. Incubate the plate at room temperature for 2 min. Then place the plate on the magnetic stand at room temperature for 5 min. i. Transfer 15 μL supernatant from the plate to the new PCR plate. 2.4 Adenylate 3’ Ends a. Add 2.5 μL of diluted A-Tailing Control (1 μL A-Tailing Control + 99 μL 9 Resuspension Buffer) to each well of the plate. b. Add 12.5 μL of A-Tailing Mix to each well of the plate. Gently pipette the entire volume up and down 10 times to mix thoroughly. c. The mixture was incubated in the following progress: 37°C for 30 min, 70°C for 5 min, hold at 4°C. 2.5 Ligate Adapters a. Add 2.5 μL of diluted Ligation Control (1 μL Ligation Control + 99 μL Resuspension Buffer) to each well of the plate. b. Add 2.5 μL of Ligation Mix to each well of the plate. c. Add 2.5 μL of RNA Adapter Index to each well of the plate. Gently pipette the entire volume up and down 10 times to mix thoroughly. d. Incubate at 30°C for 10 min. e. Add 5 μL of Stop Ligation Buffer to each well of the plate. Gently pipette the entire volume up and down 10 times to mix thoroughly. 2.5.1 Purification a. Add 42 μL of mixed AMPure XP Beads to each well of the plate. Gently pipette the entire volume up and down 10 times to mix thoroughly. Incubate the plate at room temperature for 15 min. b. Place the plate on the magnetic stand at room temperature for 15 min or until the liquid is clear. c. Remove and discard 79.5 μL supernatant from each well of the plate. d. With the plate on the magnetic stand, add 200 μL freshly prepared 80% EtOH to each well. Incubate the plate at room temperature for 30 s, and then remove and discard all of the supernatant from each well. e. Repeat step d one time for a total of two 80% EtOH washes. f. With the plate on the magnetic stand, let the samples air-dry at room temperature for 15 min. Remove the plate from the magnetic stand. g. Add 52.5 μL Resuspension Buffer to each well of the plate. Gently pipette the entire volume up and down 10 times to mix thoroughly. 10 h. Incubate the plate at room temperature for 2 min. Place the plate on the magnetic stand at room temperature for 5 minutes or until the liquid is clear. i. Transfer 50 μL supernatant from each well of the plate to the corresponding well of the new PCR plate. j. Add 50 μL of mixed AMPure XP Beads to each well of the plate. Gently pipette the entire volume up and down 10 times to mix thoroughly. Incubate the plate at room temperature for 15 min. k. Place the plate on the magnetic stand at room temperature for 15 min or until the liquid is clear. l. Remove and discard 95 μL supernatant from each well of the plate. m. With the plate on the magnetic stand, add 200 μL freshly prepared 80% EtOH to each well. Incubate the plate at room temperature for 30 s, and then remove and discard all of the supernatant from each well. n. Repeat step m one time for a total of two 80% EtOH washes. o. With the plate on the magnetic stand, let the samples air-dry at room temperature for 15 min, and then remove the plate from the magnetic stand. p. Add 22.5 μL Resuspension Buffer to each well of the plate. Gently pipette the entire volume up and down 10 times to mix thoroughly. q. Incubate the plate at room temperature for 2 min. Place the plate on the magnetic stand at room temperature for 5 min or until the liquid is clear. r. Transfer 20 μL supernatant from each well of the plate to the corresponding well of the new PCR plate. 2.6 Enrich DNA Fragments a. Add 5 μL of PCR Primer Cocktail to each well of the PCR plate. b. Add 25 μL of PCR Master Mix to each well of the PCR plate. Gently pipette the entire volume up and down to mix thoroughly. c. The mixture was incubated in the following progress: 1 cycle 98°C for 30 s; 15 cycles 98°C for 10 s, 60°C for 30 s, 72°C for 30 s; 1 cycle 72°C for 5 min; hold at 10°C. 11 2.7.1 Purification a. Add 50 μL of the mixed AMPure XP Beads to each well of the PCR plate. Gently pipette the entire volume up and down 10 times to mix thoroughly. Incubate the PCR plate at room temperature for 15 min. b. Place the plate on the magnetic stand at room temperature for 15 min or until the liquid is clear. c. Remove and discard 95 μL supernatant from each well of the PCR plate. d. With the PCR plate on the magnetic stand, add 200 μL freshly prepared 80% EtOH to each well. Incubate the PCR plate at room temperature for 30 s, and then remove and discard all of the supernatant from each well. e. Repeat step d one time for a total of two 80% EtOH washes. f. With the PCR plate on the magnetic stand, let the samples air-dry at room temperature for 15 min, and then remove the plate from the magnetic stand. g. Add 32.5 μL Resuspension Buffer to each well of the PCR plate. Gently pipette the entire volume up and down 10 times to mix thoroughly. h. Incubate the PCR plate at room temperature for 2 min. Place the PCR plate on the magnetic stand at room temperature for 5 min or until the liquid is clear. i. Transfer 30 μL supernatant from each well of the PCR plate to the corresponding well of the new PCR plate. 2.8 Validate library a. Load 1 μL sample on an Agilent Technologies 2100 Bioanalyze. b. Check the size and purity of the sample

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Data processing

1 Data preprocessing and genomic alignment Raw reads generated during high-throughput sequencing were fastq format sequences. In order to get high-quality reads that could be used for later analysis, raw reads need to be filtered by quality. Trimmomatic [1] software was first used for quality control and linker removal, and then low-quality bases and N-bases or low-quality reads were filtered out. Finally, we got high-quality clean reads. Using Rockhooper2 [2] to align clean reads to the reference genome of the experimental specie, the sample should be assessed by genomic and gene alignment
2 Genetic assembly and structural 2.1 UTR and novel gene prediction Identify the sequence of genes in an annotated gene or an unannotated area. Seeds gene sequence is a sequence area at genome that contains 10 bases at least and each one in this area are covered at least T sequence (T means a function of the2.2 Operator prediction Prokaryotes function on several related genes in are arranged in series with the often constitute manipulation substructure expression as a unit, is regulated by the common upstream area and the downstream transcription termination signal control. When transcribed, multiple structural genes are transcribed onto one mRNA strand and then translated into proteins. Rockhopper2 was used to develop the operon prediction algorithm from pure sequence features to combine experimental data of RNA-seq (ie, the gene expression quantity), that is to use bayesian classifier model to predict operons by combine the inter-genic distance and gene expression quantity, the sensitivity and specificity of the prediction algorithm are as high as 95%. Calculate and visualize 5 the predicted length distribution of operons, the number of structural genes involved, and the operon chain distribution count of genome-wide base average coverage sequence ). Through the bayesian model to judge whether the seed, seed each gene sequences repeatedly extended again after merging adjacent sequence of seeds or overlapping genes ultimately get the genetic map is obtained based on sequence alignment data reference gene annotation to the map and comparison, identification of boundary and the new genes.
3 Gene expression analysis The direct expression of a gene expression level is its abundance. The higher the degree of gene abundance, the higher the gene expression level. In transcriptome sequencing analysis, the expression level of genes was estimated by locating the count of sequencing sequence (reads) in the genome area or the exon region of the gene. The Reads count is positively correlated with the length of the gene and the depth of the sequencing, in addition to the true expression level of the gene. We used the known reference gene sequences as the database, and the expression abundance of each gene in each sample was identified by sequence similarity comparison.Rockhooper2 was used to obtain the number of reads aligned to the gene in each sample and then to calculate RPKM[3].The calculated transcript expression levels can be directly used to compare transcript expression differences between different samples, and this method can eliminate the effect of differences in transcriptional length and sequencing amount on the expression of transcripts. Visualize quantitative data to obtain box plots, density plots, expression plots, etc. to determine differences in expression across all sample
4 Difference analysis and functional analysisUsing the estimateSizeFactors function of the R package of DESeq [4] (2012) to standardize counts, and use the nbinomTest function to calculate the pvalue and foldchange of difference comparison. Pick out the difference transcripts that p-value less than 0.05 and Difference of multiples more than two, and the GO and KEGG enrichment analysis of the differential genes was performed by hypergeometric distribution tests to determine the biological functions or pathways that are mainly affected by differential genes
Assembly: GCF_003052445.1_ASM305244v1
Supplementary files format and content: counts.txt

Submission date

Mar 06, 2024

Last update date

Sep 18, 2024

Contact name

Wanbo Zhu

Organization name

Shanghai Sixth Peoples Hospital Affiliated to Shanghai Jiao Tong University

Street address

No600, Yishan road, Shanghai, China

City

Shanghai