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Sample GSM6383076

Query DataSets for GSM6383076

Status

Public on Sep 14, 2023

Title

272HMO1

Sample type

SRA

Source name

human infant feces

Organism

Bifidobacterium longum subsp. infantis

Characteristics

agent: pooled HMO
strain: ATCC 15697
treatment: 2percent pooled HMO

Treatment protocol

Bifidobacterium longum subsp. infantis were grown in mMRS media supplemented with pooled HMOs (2%, w/v), LNT (2%), LNnT (2%), NAG (2%), complex nitrogen (1% peptone, 0.5% yeast extract, 0.5% ammonium citrate) or L-cysteine (0.15%) as the nitrogen source, while 2% lactose as supplemented as the carbohydrate source.

Growth protocol

Bifidobacterium infantis was routinely propagated in De Man Rogosa Sharp medium supplemented with 0.05 % (m/v) L-cysteine hydrochloride at 37°C under anaerobic conditions.

Extracted molecule

total RNA

Extraction protocol

Bacteria cells were harvested at exponential phase and pelleted immediately by centrifugation at 12,000 × g for 1 min and resuspended in 1 ml of RNAlater Ambion (Thermo Fisher Scientific, Waltham, MA, USA), and incubated at 4 °C overnight and then stored at – 80 °C until further analysis. RNA isolation was performed using a Ambion RNAqueousTM kit (Thermo Fisher Scientific) following manufacturer instructions. An additional step of bead-beating for cell disruption (FastPrep-24TM 5G, MP Biomedicals Inc, Santa Ana, CA, USA) was added to the RNA isolation procedure. Total RNA was immediately treated with the Turbo DNase free kit (Ambion, Thermo Fisher Scientific).
Total RNA was treated with Ribo-Zero rRNA Removal kit, Bacteria (Illumina, San Diego, CA, USA) to remove ribosomal RNA according to manufacturer instructions. The depleted RNA was purified with RNAeasy MinElute Cleanup kit (Qiagen, Valencia, CA, USA). Whole transcriptome libraries were constructed using TruSeq Stranded mRNA Library Preparation Kit (Illumina) or NEBNext Ultra II Directional RNA library Prep Kit for Illumina (New England Biolabs Inc, MA, USA) following manufacturer instructions.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NextSeq 500

Description

UMA272 HMO as N.txt
DESeq2-UMA272.299.302nagctrl.nagcys.summary.csv

Data processing

The raw reads quality was checked by FastQC (v1.0.0).
For libraries prepared by NEBNext Ultra II Directional RNA library Prep Kit, the sequencing adapters were removed by Trimmomatic (v0.32). For libraries prepared by TruSeq Stranded mRNA Library Preparation Kit, the sequencing adapters were trimmed by using the Illumina FASTQ generation pipelines.
The quality-controlled and trimmed reads were then aligned to the Bifidobacterium longum subsp. infantis ATCC 15697T genome (NCBI accession number: NC_011593.1) using bowtie2 (v2.1.0)
The aligned reads were sorted by SAMtools (v1.0) and counted against specific genomic locus by using HTSeq (v0.6.1) for differential expression analysis.
Differentially expressed gene (DEG) analysis was performed using the R Package DESeq2 (v3.3.1) and log2 fold changes obtained for one condition relative to other
Assembly: Bifidobacterium longum subsp. infantis ATCC 15697
Supplementary files format and content: Matrix table with raw gene counts for every gene and every sample
Supplementary files format and content: Normalized abundance measurements from DeSeq2

Submission date

Jul 25, 2022

Last update date

Sep 14, 2023

Contact name

Shuqi Li

Organization name

Harvard Medical School and Brigham and Women's Hospital

Department

Neurology

Lab

Cox Lab