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Sample GSM5400862

Query DataSets for GSM5400862

Status

Public on Aug 18, 2022

Title

Mir_B

Sample type

SRA

Source name

Blank

Organism

blank sample

Characteristics

sample type: PBS
tissue: none

Treatment protocol

Endometrial fluid was aspirated with a catheter used for embryo transfer (Frydman, Instrumentos Médicos Estériles SA, Spain) connected to a 10 mL syringe, under abdominal ultrasound guidance. Sample extraction was performed by gentle manual application of negative pressure with the syringe. To prevent contamination with cervical mucus, aspiration was interrupted at the internal cervical os. Aspirate volumes ranged from 5 uL up to 50 uL. After the aspiration, the 10 mL syringe was replaced with a 2 mL syringe containing 1.5 mL saline solution and the aspirates were mixed and expelled in a cryogenic tube (5-50 uL of EF + 1500 uL 1XDPBS (Gibco, Thermo Fisher Scientific #14190250). The mixed samples were centrifuged to remove contaminants at 2,000 x g for 5 min and the supernatants were then frozen at −80 °C until processed. The dilution of the supernatants was 1:30 and they had a volume comprised between 400uL and 1300uL. Extracellular vesicle enrichment was done with Total Exosome Isolation Reagent from cell culture media (Invitrogen by ThermoFisher Scientific, #4478359). The EF supernatants were centrifuged at 3000 g for 30 min at 4 ºC, then 400uL were transferred to a fresh tube and 1 volume of the Total Exosome Isolation Reagent (1:1) was added. The mixture was incubated for 30 min at room temperature and then centrifuged at 10,000 g for 1 hour at 4 ºC. The supernatant was discarded and the EVs that were in the pellet were resuspended in 100 uL of 1 x DPBS.

Extracted molecule

total RNA

Extraction protocol

Total RNA from extracellular vesicles from endometrial fluid was extracted using either the mirVana PARIS kit (Ambion) or the Plasma/Serum RNA Purification kit (Norgen), as detailed in the SAMPLES section
Sequencing libraries were prepared following the protocol included with the kit “NEXTflex™ Small RNA-Seq Kit v3,” (©Bioo Scientific Corp. Catalog #5132-05).

Library strategy

ncRNA-Seq

Library source

transcriptomic

Library selection

size fractionation

Instrument model

Illumina HiSeq 2500

Description

mirVana PARIS kit (Ambion)

Data processing

FASTQs were trimmed for the adapters following the recommendations of the NEXTflex™ Small RNA-Seq Kit manufacturers.
We used Bowtie 1.0.1 to align the reads against the human genome (GRCh38), with a mismatch 0, to avoid false positives. We select mirbase v22 to quantify mature micrornas employing Partek Flow application, software version 7.0. Only the miRNAs with 10 or more reads were considered.
Mapped reads were quantified to GENCODE-v26 using Partek E/M method, so the processed data file contains counts values normalized by Partek E/M method
Genome_build: GRCh38
Supplementary_files_format_and_content: read count table

Submission date

Jun 25, 2021

Last update date

Aug 18, 2022

Contact name

Ana Maria Aransay

E-mail(s)

amaransay@cicbiogune.es

Phone

0034944061325

Organization name

CIC bioGUNE