characteristics.text: ERC000011
characteristics.characteristic: ENA-CHECKLIST
characteristics.text: 2022-10-24T12:31:17Z
characteristics.characteristic: ENA-FIRST-PUBLIC
characteristics.text: 2023-04-01T00:23:04Z
characteristics.characteristic: ENA-LAST-UPDATE
characteristics.text: SAMEA110448178
characteristics.characteristic: External Id
characteristics.text: Latvian Biomedical Research and Study Centre
characteristics.characteristic: INSDC center name
characteristics.text: 2022-10-24T12:31:17Z
characteristics.characteristic: INSDC first public
characteristics.text: 2023-04-01T00:23:04Z
characteristics.characteristic: INSDC last update
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS12546208
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-12014:BCP-32_1
characteristics.characteristic: Submitter Id
characteristics.text: ArrayExpress
characteristics.characteristic: broker name
characteristics.text: human
characteristics.characteristic: common name
characteristics.text: Protocols: BC patients were recruited between June 2019 and October 2020 at Riga East University Hospital and followed-up 18 months after the surgery. Inclusion criteria: previously untreated invasive primary breast cancer diagnosed by core needle biopsy, stage II to III at diagnosis, age 18-78 years, prescribed NAC. Exclusion criteria: blood transfusion in the last six months, another oncological disease. The blood samples were collected in specified time points - at the time of diagnosis, at the end of NAC, 7 days after the surgery and 6 and 12 after the surgery. Blood samples were collected in EDTA-coated tubes and processed at room temperature within 2 hours. Plasma samples were centrifuged twice at 3000g for 10 min, aliquoted and stored at -80°C. BC and adjacent normal tissue specimens were macroscopically dissected by a histopathologist during surgery and stored in RNALater (Applied Biosystems, USA) at -20°C till processing. The NAC regimens contained Doxorubicin, Docetaxel, Cyclophosphamine, Paclitaxel, 5FU and Epirubicin. EVs were isolated from 1 ml of plasma, using size exclusion chromatography (SEC). SEC columns were prepared from 10 ml of Sepharose CL2B (Cytiva, USA) in TELOS SPE columns (Kinesis, USA). Plasma samples were loaded on the columns and gravity-eluted with PBS-DEPC, and the eluate was collected in 15 sequential 500 μl fractions. Fractions were measured with Zetasizer Nano ZS (Malvern, UK) and fractions containing particles larger than 35 nm, were collected and concentrated to 100 μl using Amicon Ultra 3 kDa centrifugal filters (Merck Millipore, Germany). To check the purity and quality, EVs from 4 patients and controls were analyzed by transmission electron microscopy (TEM) as described before (Endzeliņš et al., 2017). Before RNA extraction, the EV samples were treated with 1 mg/ml proteinase K (Thermo Fisher Scientific, USA) for 30 min at + 37˚C. Proteinase was inactivated by heating the sample at + 65˚C for 10 minutes, and then the samples were treated with 10 ng/μl RNAse A (Thermo Fisher Scientific, USA) for 15 minutes at +37˚C. Immediately after that, EV samples were lysed by adding 5 volumes of QIAzol Lysis reagent and EV-RNA was extracted using miRNeasy Micro Kit (Qiagen, USA) according to the manufacturer’s protocol. Briefly, 1 ml of chloroform was added to the EV lysate, incubated at room temperature for 10 minutes and centrifuged for 15 minutes at 12000 g at +4˚C. The aqueous phase was transferred to fresh tube and 1.5 vol of 100% ethanol was added, and the mixture was loaded onto MinElute Spin column. Columns were centrifuged and washed according to manufacturer’s suggestions and the RNA was eluted using 12 μl of RNAse-free water. To determine the concentration and assess the quality, the RNA was measured using Agilent 2100 Bioanalyzer and RNA 6000 Pico Kit (Agilent technologies, USA).From the tissue specimens, approximately 20 mg of tissues were homogenized using QIAzol Lysis Reagent (Qiagen, USA) and Lysing Matrix A tubes in a FastPrep-24 homogenizer (MP Biomedicals, USA). RNA was isolated using miRNeasy Micro Kit (Qiagen) following the small RNA enrichment protocol provided by the manufacturer, thus obtaining both, the long and small RNA fraction of each sample. EV-RNA libraries were constructed from half of the entire yield of extracted EV RNA without any size separation steps, whereas tissue small RNA libraries were constructed from 10 ng of tissue small RNA fraction using CleanTag® Small RNA Library Prep Kit (Trilink Biotechnologies, USA). The libraries were cleaned using Blue Pippin DNA Size Selection with 3% gel Blue Pippin Cassette (Sage Science, USA) setting target length to 130 - 250 bp. Transcriptome libraries were built from 100 ng of long tissue RNA fraction using TruSeq Stranded mRNA library Prep (Illumina, USA) following manufacturer's instructions. Libraries were size-selected using Blue Pippin system with 2% gel Blue Pippin Cassette (Sage Science, USA) with a range of 200 - 600 bp and their quality and quantity was assessed using Agilent DNA kit (Agilent technologies, USA).
characteristics.characteristic: description
characteristics.text: adult
characteristics.characteristic: developmental stage
characteristics.text: breast cancer
characteristics.characteristic: disease
characteristics.text: extracellular vesicles
characteristics.characteristic: fraction
characteristics.text: BCP-32
characteristics.characteristic: individual
characteristics.text: Homo sapiens
characteristics.characteristic: organism
characteristics.text: Homo sapiens
characteristics.characteristic: organism
characteristics.text: blood plasma
characteristics.characteristic: organism part
characteristics.text: Homo sapiens
characteristics.characteristic: scientific_name
characteristics.text: female
characteristics.characteristic: sex
characteristics.text: BCP-32_1
characteristics.characteristic: title