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Sample GSM3585542

Query DataSets for GSM3585542

Status

Public on Mar 26, 2019

Title

Reversal reaction leprosy skin biopsy 5

Sample type

SRA

Source name

skin

Organisms

Mycobacterium leprae; Homo sapiens

Characteristics

diagnosis: Reversal Reaction Leprosy (RR)
age: 53
host gender: Female
tissue: skin

Extracted molecule

total RNA

Extraction protocol

Six millimeter punch biopsies were taken near the border of the lesion and specimens were embedded in OCT medium (Ames, Elkhar, IN) then flash frozen in liquid nitrogen. Frozen tissue sections of nine L-lep, nine RR, and six T-lep frozen skin lesions were cut into multiple ten micron sections and lysed in Qiagen RLT buffer with 0.1% b-mercaptoethanol (Invitrogen). To ensure efficient lysis of mycobacterial cell wall, lysates were homogenized with 0.1mm silica beads (MP Biomedicals, Santa Ana, CA) in the FastPrep3000 twice at a setting of 6.5 for 45s. RNA was extracted via Qiagen AllPrep Kit (Qiagen, Hilden, Germany).
Ribosomal RNA was depleted with the Illumina Ribo-Zero Gold rRNA Removal Kit (Epidemiology) before sequencing library preparation with the Illumina Truseq Stranded Total RNA Sample Preparation Kit. Libraries were sequenced on the HiSeq2000 Sequencer (Illumina) at single-end 50 bp reads.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Description

RR5

Data processing

Reads with poor quality were trimmed by Trim Galore! (http://www.bioinformatics.babraham.ac.uk/projects/trim_galore) and high quality reads were mapped to the hg19 human genome via STAR (Dobin et al., 2013). Unmapped reads after human genome alignment were mapped to the Br4923 M. leprae genome (Assembly ASM2668v1). The sum of exonic reads per gene were counted using HTSeq (Anders) using the GENCODE GRCh37-mapped version Release 24 (Harrow et al., 2012). Although, M. leprae 23S was calculated by a 96bp region (1343455-1343551).
Genome_build: human hg19 and Br4923 M. leprae genome (Assembly ASM2668v1)
Supplementary_files_format_and_content: Filtered exonic read counts (containing genes with ≥ 5 reads in at least one patient group) from bacterial and human transcriptomes were normalized via DESeq2 (Love et al., 2014) using default parameters, except for basing scaling factors on only human genes. DESeq2 normalized counts per M. leprae gene or Human gene were output for processed expression files

Submission date

Jan 31, 2019

Last update date

Mar 27, 2019

Contact name

Dennis Jay Montoya

E-mail(s)

[email protected]

Organization name

UC Davis School of Medicine

Department

Biochemistry and Molecular Medicine