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| Status |
Public on Oct 09, 2021 |
| Title |
AD_HN4_rep2 |
| Sample type |
SRA |
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| Source name |
Geobacter sulfurreducens
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| Organism |
Geobacter sulfurreducens KN400 |
| Characteristics |
strain: KN400
treatment: with HN4+ addition
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| Growth protocol |
Three electrodes of two-chambered H-shaped cells with a liquid volume of 60 mL and a headspace volume of 90 mL in each chamber were assembled as previously described (Jing et al. 2019). The anode and cathode were made of graphite plates (2 × 3 × 0.3 cm) (JingLong Special Carbon, Beijing, China), which were polished using sandpaper (grit type 400), sonicated for 30 mins, soaked in 1 M HCl overnight and then rinsed at least five times with Milli-Q water. A saturated calomel electrode (SCE) was used as the reference electrode. The electrolyte was freshwater medium (FWNN medium) buffered with carbonate as previously reported (Nevin et al. 2009), except that the anolyte was supplied with 12 mM sodium acetate. To perform the BNF test, NH4Cl was eliminated from the anolyte. The reactor was autoclaved at 121 °C for 30 min and then purged with sterile N2-CO2 (80:20) for at least 30 min to remove oxygen and to saturate N2. Geobacter sulfurreducens strain KN400 was initially cultured in N-deficient NBAF medium (without NH4Cl) as previously reported (Liu et al. 2019). To start the process, 5 mL of bacterial cells in logarithmic phase was inoculated into the anode chamber, in which the anode was polarized at 0.3 V (vs. SCE) with a multichannel electrochemical workstation (CHI 1000 C, Shanghai, China). The current was recorded continuously and normalized to the maximum projected area of the anode. All experiments were conducted in triplicate at 30 °C, and the mean values were reported.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Cells on the anode were collected at the mid-log phase, and were flash frozen in liquid nitrogen then stored at -80C until needed. The total RNA was extracted using TRIzol reagent (Invitrogen, California, USA) as previously described (Liu et al. 2021)
A total amount of 3 μg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer’s recommendations and index codes were added to attribute sequences to each sample.Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer(5X). First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase(RNase H-). Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3’ ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were ligated to prepare for hybridization. Then USER Enzyme (NEB, USA) was ued to degrade the second strand of cDNA containing U, In rder to select cDNA fragments of preferentially 250~300 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, PCR products were purified (AMPure XP system) and ibrary quality was assessed on the Agilent Bioanalyzer 2100 system.
The clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumia) according to the manufacturer’s instructions. After cluster generation, the library preparations were sequenced on an Illumina Novaseq platform and 150 bp paired-end reads were generated.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
Quality control. Raw data (raw reads) of fastq format were firstly processed through in-house perl scripts. In this step, clean data (clean reads) were obtained by removing reads containing adapter, reads containing ploy-N and low Reads mapping to the reference genome . quality reads from raw data. At the same time, Q20, Q30 and GC content the clean data were calculated. All the downstream analyses were based on the clean data with high quality.
Reads mapping to the reference genome. Reference genome and gene model annotation files were downloaded from genome website directly. Both building index of reference genome and aligning clean reads to reference genome were used Bowtie2-2.2.3.
Novel gene and gene structure analysing. Rockhopper was used to identify novel genes, operon, TSS, TTS and Cis-natural antisense transcripts. It can be used for efficient and accurate analysis of bacterial RNA-seq data, and that it can aid with elucidation of bacterial transcriptomes. Then, we extract upstream 700bp sequence of Transcription Start Site for predicting promoter using TDNN.(Time-DelayNeural Network)
Quantification of gene expression level.HTSeq v0.6.1 was used to count the reads numbers mapped to each gene. And then FPKM of each gene was calculated based on the length of the gene and reads count mapped to this gene. FPKM, expected number of Fragments Per Kilobase of transcript sequence per Millions base pairs sequenced, considers the effect of sequencing depth and gene length for the reads count at the same time, and is currently the most commonly used method for estimating gene expression levels
Differential expression analysis. (For DESeq with biological replicates) Differential expression analysis of two conditions/groups (two biological replicates per condition) was performed using the DESeq R package (1.18.0). DESeq provide statistical routines for determining differential expression in digital gene expression data using a model based on the negative binomial distribution. The resulting P-values were adjusted using the Benjamini and Hochberg’s approach for controlling the false discovery rate . Genes with an adjusted P-value <0.05 found by DESeq were assigned as differentially expressed. (For edgeR without biological replicates) Prior to differential gene expression analysis, for each sequenced library, the read counts were adjusted by edgeR program package through one scaling normalized factor. Differential expression analysis of two conditions was performed using the edgeR R package (3.22.5). the P values were adjusted using the TMM(trimmed mean of M-valucted P-value of 0.05 and log2(Fold change) of 1 were set as the threshold for significantly differential expression.
Genome_build: https://www.ncbi.nlm.nih.gov/nuccore/CP002031.1
Supplementary_files_format_and_content: tab-delimited text files include FPKM values for each Sample
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| Submission date |
Oct 06, 2021 |
| Last update date |
Oct 09, 2021 |
| Contact name |
jing xian yue |
| E-mail(s) |
[email protected]
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| Phone |
18150817630
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| Organization name |
Fujian Agriculture and Forestry University
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| Street address |
No.15 Shangxiadian Road
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| City |
Fuzhou |
| ZIP/Postal code |
350002 |
| Country |
China |
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| Platform ID |
GPL30823 |
| Series (1) |
| GSE185414 |
Anode respiration-dependent biological nitrogen fixation by Geobacter sulfurreducens |
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| Relations |
| BioSample |
SAMN22082209 |
| SRA |
SRX12498512 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5614418_AD_NH4_2.txt.gz |
150.9 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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