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Sample GSM5116148

Query DataSets for GSM5116148

Status

Public on Sep 28, 2021

Title

Urine_Fe_12mo_rep6

Sample type

SRA

Source name

Urine

Organism

Rattus norvegicus

Characteristics

gender: Male
strain: Sprague-Dawley
metal: Fe
time: 12mo

Treatment protocol

Pellets were surgically implanted into the gastrocnemius muscle, and each animal received 2 pellets per limb for a total of 4 pellets per rat. Prior to use, pellets were cleaned and chemically sterilized as previously described (https://doi.org/10.1177%2F1091581814565038)

Growth protocol

Male Sprague–Dawley rats (Rattus norvegicus; age, approximately 30 d; weight, 75 to 100 g) were purchased from Envigo (Barrier 208A, Frederick, MD). Rats were allowed to acclimate in the AFRRI vivarium for at least 2 weeks prior to the start of experiments. The room was maintained at standard temperature (21 ± 2°C) and humidity (30% to 70%), with a 12:12-h light:dark cycle (lights on, 0600) and access to standard rat chow (Teklad Global Rodent Diet 8604, Envigo) and water ad libitum. Rats were pair-housed in plastic microisolation cages (23.8 × 45.4 cm) with filter tops and bedding (Teklad Sani-Chips, Envigo), changed 2 or 3 times weekly. All procedures involving animals were conducted to achieve maximal possible wellbeing of the rats, were IACUC-approved (protocol no. 2016-05-006) prior to the start of the study, and were performed in compliance with the guidelines set forth in the Guide for the Care and Use of Laboratory Animals in an AAALAC-accredited facility.

Extracted molecule

total RNA

Extraction protocol

Total RNA was isolated from urine samples using the Urine Cell-Free Circulating RNA Purification Mini Kit (Cat. 56900, Norgen Biotek, Thorold, ON, Canada) following manufacturer's protocol. Briefly, 2 ml of cell-free urine was mixed with Binding Solution K and transferred to spin column. RNA within the sample was bound to the spin column while the urine was discarded as flow-through. Following application of Lysis Buffer A, the flow-through was precipitated with isopropanol and subjected to repeat centrifugation through the column to allow maximum RNA binding. The column was then washed twice with Wash Solution A, centrifuged to remove left over alcohol, and transferred to a new RNAse-free tube for elution. The concentration of total RNA was assessed with the Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA).
Library preparation and sequencing was performed by Norgen Biotek. Briefly, library preparation was done using the Norgen Biotek Small RNA Library Prep Kit (Cat. 63600). Sequencing was performed using the NextSeq 500/550 High Output Kit v2 (51 Cycles using a 75-Cycle Kit) on the Illumina NextSeq 500 platform.

Library strategy

miRNA-Seq

Library source

transcriptomic

Library selection

size fractionation

Instrument model

Illumina NextSeq 500

Data processing

Single-end reads for miRNA were mapped to miRbase version 21 using BowTie aligner with the following parameters: Number of Mismatches: 2, Length of the Seed: 20, Alignment Type: -n, Maximum Allowed Multiple Mappings: 10. The alignment was performed by Norgen Biotek using sRNAbench and sRNAtoolbox.
Supplementary_files_format_and_content: Tab-delimited matrix table with raw read counts for every miRNA

Submission date

Mar 01, 2021

Last update date

Sep 28, 2021

Contact name

Yuan Wen

E-mail(s)

ywen2@g.uky.edu

Phone

8592186846

Organization name

University of Kentucky