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Sample GSM6428577

Query DataSets for GSM6428577

Status

Public on Sep 23, 2022

Title

03idBALBCmo2rp3

Sample type

SRA

Source name

Lacrimal Gland

Organism

Mus musculus

Characteristics

tissue: Lacrimal Gland
strain: BALB/cJ
age: 2 months
Sex: male

Growth protocol

Mice were fed ad libitum and kept under a 12-hour light/12-hour dark cycle

Extracted molecule

total RNA

Extraction protocol

Each lacrimal gland was transferred into 2mL bead beating tubes (cat# 19-628, OMNI, Kennesaw GA) containing 700 µL of Qiazol lysis reagent (#79306, Qiagen). The lacrimal gland was disrupted using Bead Ruptor 4 (cat# 25-010, Omni) in two cycles: first at speed 5, for 40 sec and then at speed 5, for 30 sec. Between the two cycles, the tubes were placed on ice for 2 mins. RNA was isolated with the miRNeasy Mini kit (#217084, Qiagen) including a DNAse treatment step, according to the manufacturer’s instructions.
The amount of total RNA was estimated by a Nanodrop ND-1000 spectrophotometer and checked for purity and integrity (RIN) in a Bioanalyzer-2100 device (Agilent Technologies, Inc., Santa Clara, CA). 800 ng of total RNA (RIN>8) from each sample was used to prepare RNAseq libraries using the NEBNext rRNA Depletion Kit (Human/Mouse/Rat) followed by the NEBNext Ultra II RNA Library Prep Kit for Illumina (9 cycles of PCR). Completed libraries had been sequenced (2 x 75 paired ends) using Illumina NextSeq500 to generate 50M reads for each sample.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NextSeq 500

Data processing

Data was analyzed by ROSALIND® (https://rosalind.bio/), with a HyperScale architecture developed by ROSALIND, Inc. (San Diego, CA).
Reads were trimmed using cutadapt
Quality scores were assessed using FastQC
Reads were aligned to the Mus musculus genome build GRCm38 using STAR
Individual sample reads were quantified using HTseq
Reads were normalized via Relative Log Expression (RLE) using DESeq2 R library
Read Distribution percentages, violin plots, identity heatmaps, and sample MDS plots were generated as part of the QC step using RSeQC
DEseq2 was also used to calculate fold changes and p-values and perform optional covariate correction. Clustering of genes for the final heatmap of differentially expressed genes was done using the PAM (Partitioning Around Medoids) method using the fpc R library
Assembly: Mus musculus genome build GRCm38
Supplementary files format and content: Raw Count matrix are provided in .txt file
Supplementary files format and content: Normalized Count matrix are provided in .txt file

Submission date

Aug 02, 2022

Last update date

Sep 23, 2022

Contact name

Helen Makarenkova

E-mail(s)

hmakarenk@scripps.edu

Organization name

The Scripps Research Institute

Department

Molecular Medicine