title: B09
organism: Homo sapiens
description: Protocols: The NSq, BE, LGD tissue samples were collected at endoscopies performed at St Vincents Hospital, Sydney, Australia. These were obtained from patients prospectively enrolled in an Australia-wide research collaboration entitled PROBE-NET: Progression of Barretts Esophagus to Esophageal Adenocarcinoma Network. The EAC specimens were either obtained from patients at St Vincents Hospital or the Department of Surgery at Flinders University, Adelaide, Australia. All cancer specimens were pre-treatment, biopsies from radio-chemotherapy naive patients. All tissue samples were collected from the tubular esophagus at or above the gastroesophageal junction, which was defined as the proximal border of the gastric rugal folds. In patients with BE or LGD, a columnar mucosal segment of at least 1 cm was required for inclusion in the present study. Prior to RNA isolation, histologic sections of all tissue specimens were reviewed by three experienced GI-pathologists to verify the correct diagnosis and determined tissue cellularity. Samples had to contain a minimum of 50% diagnosis defining specialized epithelium to be deemed suitable for RNA extraction. RNA extraction and quality control prior to cDNA library construction After tissue-homogenization using a Polytron Homogenizer PT 3100 (Thomas Scientific, Swedesboro, NJ) RNA extraction was performed using the QIAGEN RNeasy Mini Kit, (Cat #74104, QIAGEN, Valencia, CA) following the manufacturers protocol. RNA yield and quality was measured using a BioSpec nano spectrophotometer (Shimadzu Scientific Instruments, Sydney, Australia) and integrity was verified on a Bioanalyzer separation chip (Agilent Technologies, Forest Hill, Victoria, Australia). 260/280 ratios were > 2.0 for all cases and a minimum RNA Integrity Number (RIN) of 6 required for samples to be taken forward to library preparation for RNA sequencing. Median RNA yield per sample was 5.9 g (IQR 3.3 10.5g) with a median amount of RNA sequenced per sample being 3.3 g (2.1 4.0g). 500ng of total RNA was used as input material for library preparation using the TruSeq Stranded Total RNA Sample Prep Kit (Illumina, USA) according to manufacturers instructions. Individual libraries were indexed as recommended by Illumina. Indexed DNA libraries were analyzed individually using an Agilent Technologies 2100 Bioanalyzer with the DNA 1000 kit according to the manufacturers instructions (Agilent Technologies, USA). Libraries were diluted and pooled to a final concentration of 10 nM each in nuclease-free H2O (Ambion, USA). Pooled libraries were quantitated using a Life Technologies Qubit 2.0 Fluorometer with the Qubit dsDNA HS Assay Kit (Life Technologies, USA) and further diluted to 2 nM. Final DNA library concentration was confirmed using a Qubit dsDNA HS Assay Kit. PCR-competent library DNA concentration was verified using the universal KAPA Library Quantification Kit for Illumina Sequencing Platforms according to manufacturers instructions (KAPA Biosystems, USA). An Applied Biosystems 7900HT Fast Real-Time PCR machine (Life Technologies, USA) was used for quantitative real-time PCR.
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2017-07-23T17:01:23Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2018-03-09T09:27:03Z
attributes.tag: External Id
attributes.value: SAMEA3670218
attributes.tag: INSDC center name
attributes.value: Garvan Institute of Medical Research
attributes.tag: INSDC first public
attributes.value: 2017-07-23T17:01:23Z
attributes.tag: INSDC last update
attributes.value: 2018-03-09T09:27:03Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-4054:B09
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: common name
attributes.value: human
attributes.tag: disease staging
attributes.value: Barrett's
attributes.tag: organism part
attributes.value: esophagus tissue
attributes.tag: sample name
attributes.value: E-MTAB-4054:B09
attributes.tag: scientific_name
attributes.value: Homo sapiens