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| Status |
Public on Sep 16, 2020 |
| Title |
SL rep2 |
| Sample type |
SRA |
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| Source name |
Fermented milk
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| Organisms |
Levilactobacillus brevis; Streptococcus thermophilus ASCC 1275 |
| Characteristics |
strain: NPS-QW-145
fermentation time: 18 h
treatment: lactic acid bacteria
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated and purified using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer's procedure. The RNA amount and purity of each sample was quantified using NanoDrop ND-1000 (NanoDrop, Wilmington, DE, USA). The RNA integrity was assessed by Agilent 2100 with RIN number >7.0.
Approximately 5 ug of total RNA was used to deplete ribosomal RNA according to the manuscript of the Ribo-Zero™ rRNA Removal Kit (Illumina, San Diego, USA). After removing ribosomal RNAs, the left RNAs were fragmented into small pieces using divalent cations under high temperature. Then the cleaved RNA fragments were reverse-transcribed to create the cDNA,which were next used to synthesise U-labeled second-stranded DNAs with E. coli DNA polymerase I, RNase H and dUTP.. An A-base is then added to the blunt ends of each strand, preparing them for ligation to the indexed adapters. Each adapter contains a T-base overhang for ligating the adapter to the A-tailed fragmented DNA. Single- or dual-index adapters are ligated to the fragments, and size selection was performed with AMPureXP beads. After the heat-labile UDG enzyme treatment of the U-labeled second-stranded DNAs,The ligated products are amplified with PCR by the following conditions: initial denaturation at 95℃ for 3 min; 8cycles of denaturation at 98℃ for 15 sec, annealing at 60℃ for 15 sec, and extension at 72℃ for 30 sec; and then final extension at 72℃ for 5 min. The average insert size for the final cDNA library was 300 bp (±50 bp).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 4000 |
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| Data processing |
First, sequencing adapters were removed from sequencing reads using cutadapt v1.9. Secondly, low quality reads were trimmed by fqtrim v0.95 using a sliding-window algorithm. Thirdly, reads were aligned to the host genome using bowtie2 v2.2.0 to remove host contamination. Once quality-filtered reads were obtained, they were de novo assembled to construct the Metatrascriptome for each sample by Trinityv2.2.0.
All Metatrascriptome contigs of all samples were clustered by CD-HIT v4.6.1 to obtain unigenes. Unigene abundance for a certain sample were estimated by TPM based on the number of aligned reads by bowtie2 v2.2.0. The lowest common ancestor taxonomy of unigenes were obtained by aligning them against the NCBI NR database by DIAMOND v 0.7.12. Similarly, the functional annotation (GO, KEGG, eggnog, CAZy, CARD, PHI) of unigenes were obtained. Based on the taxonomic and functional annotation of unigenes, along with the abundance profile of unigenes, the differential analysis were carried out at each taxonomic or functional or gene-wise level by Fisher's exact test (non-replicated groups) or Kruskal Wallis test (replicated groups).
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| Submission date |
Sep 15, 2020 |
| Last update date |
Sep 16, 2020 |
| Contact name |
Tingting Xiao |
| E-mail(s) |
u3006403@connect.hku.hk
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| Organization name |
University of Hong Kong
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| Street address |
Kadoorie Building, Pokfulam Road, Hong kong Island
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| City |
Hong Kong |
| ZIP/Postal code |
999077 |
| Country |
China |
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| Platform ID |
GPL29153 |
| Series (1) |
| GSE157976 |
Meta-transcriptomic Analyses Reveal Improved Gamma-amino butyric acid Production Machinery in Levilactobacillus brevis NPS-QW 145 Co-cultured with Streptococcus thermophilus ASCC1275 during Milk Fermentation |
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| Relations |
| BioSample |
SAMN16134433 |
| SRA |
SRX9125170 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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