Dataset Information

RNAseq_HGSOC_marker_paper - samples

ABSTRACT: Somatic RNA for 37 samples was extracted using the Qiagen Qiasymphony RNA protcol (cat no 931636). The tissue was initially homogenised using a Qiagen Bioruptor, followed by the manufacturers recommended protocol (including DNase digestion). The resulting RNA the underwent quality control as follows: firstly, A260 and A280nm were measured on a Denovix DS-11 Fx to qualitatively illustrate A260/280nm and A260/230nm ratios as measures of RNA purity. A260/280 had to be 2.0 and A260/230 had to be 2.0-2.2. Then RNA was quantified using LifeTechnologies Qubit RNA BR kit (cat no Q10210). RNAseq was carried out by the Edinburgh Clinical Research Facility on an Illumina NExtSeq500. Total RNA samples were assessed on the Agilent Bioanalyser (Agilent Technologies, #G2939AA) with the RNA 6000 Nano Kit (#5067-1512) for quality and integrity of total RNA, and then quantified using the Qubit 2.0 Fluorometer (Thermo Fisher Scientific Inc, #Q32866) and the Qubit RNA HS assay kit (#Q32855). Libraries were prepared from total-RNA sample using the NEBNext Ultra 2 Directional RNA library prep kit for Illumina (#E7760S) with the NEBNext rRNA Depletion kit (#E6310) according to the provided protocol. 400ng of totalRNA was then added to the ribosomal RNA (rRNA) depletion reaction using the NEBNext rRNA depletion kit (Human/mouse/rat) (#E6310). This step uses specific probes that bind to the rRNA in order to cleave it. rRNA-depleted RNA was then DNase treated and purified using Agencourt RNAClean XP beads (Beckman Coulter Inc, #66514). RNA was then fragmented using random primers before undergoing first strand and second strand synthesis to create cDNA. cDNA was end repaired before ligation of sequencing adapters, and libraries were enriched by PCR using the NEBNext Multiplex oligos for Illumina set 1 and 2 (#E7500). Final libraries had an average peak size of 271bp. Libraries were quantified by fluorometry using the Qubit dsDNA HS assay and assessed for quality and fragment size using the Agilent Bioanalyser with the DNA HS Kit (#5067-4626). Sequencing was performed using the NextSeq 500/550 High-Output v2 (150 cycle) Kit (# FC- 404-2002) on the NextSeq 550 platform (Illumina Inc, #SY-415-1002). Libraries were combined in an equimolar pool based on the library quantification results and run across 5 High-Output Flow Cell v2.5.

PROVIDER: EGAD00001010139 | EGA |

REPOSITORIES: EGA

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Project description:Somatic RNA for 40 samples matched to the WGS was extracted using the Qiagen Qiasymphony RNA protcol (cat no 931636). The tissue was initially homogenised using a Qiagen Bioruptor, followed by the manufacturers recommended protocol (including DNase digestion). The resulting RNA the underwent quality control as follows: firstly, A260 and A280nm were measured on a Denovix DS-11 Fx to qualitatively illustrate A260/280nm and A260/230nm ratios as measures of RNA purity. A260/280 had to be 2.0 and A260/230 had to be 2.0-2.2. Then RNA was quantified using LifeTechnologies Qubit RNA BR kit (cat no Q10210). RNAseq was carried out by the Edinburgh Clinical Research Facility on an Illumina NExtSeq500. Total RNA samples were assessed on the Agilent Bioanalyser (Agilent Technologies, #G2939AA) with the RNA 6000 Nano Kit (#5067-1512) for quality and integrity of total RNA, and then quantified using the Qubit 2.0 Fluorometer (Thermo Fisher Scientific Inc, #Q32866) and the Qubit RNA HS assay kit (#Q32855). Libraries were prepared from total-RNA sample using the NEBNext Ultra 2 Directional RNA library prep kit for Illumina (#E7760S) with the NEBNext rRNA Depletion kit (#E6310) according to the provided protocol. 400ng of totalRNA was then added to the ribosomal RNA (rRNA) depletion reaction using the NEBNext rRNA depletion kit (Human/mouse/rat) (#E6310). This step uses specific probes that bind to the rRNA in order to cleave it. rRNA-depleted RNA was then DNase treated and purified using Agencourt RNAClean XP beads (Beckman Coulter Inc, #66514). RNA was then fragmented using random primers before undergoing first strand and second strand synthesis to create cDNA. cDNA was end repaired before ligation of sequencing adapters, and libraries were enriched by PCR using the NEBNext Multiplex oligos for Illumina set 1 and 2 (#E7500). Final libraries had an average peak size of 271bp. Libraries were quantified by fluorometry using the Qubit dsDNA HS assay and assessed for quality and fragment size using the Agilent Bioanalyser with the DNA HS Kit (#5067-4626). Sequencing was performed using the NextSeq 500/550 High-Output v2 (150 cycle) Kit (# FC- 404-2002) on the NextSeq 550 platform (Illumina Inc, #SY-415-1002). Libraries were combined in an equimolar pool based on the library quantification results and run across 5 High-Output Flow Cell v2.5.

Project description:Purpose: Pulmonary tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), has long been associated with the development of lung adenocarcinoma (LUAD) and sarcoidosis. These chronic lung diseases share histopathological similarities that challenge differential diagnosis. Methods: Human lung tissues were used for total RNA extraction with RNeasy Plus Mini Kit (Qiagen) according to the manufacturer’s protocol. RNA purity was assessed using a Nanodrop ND-2000 (Thermo Scientific), and each RNA sample had an A260:A280 ratio above 1.8 and an A260:A230 ratio above 2.0. RNA integrity was evaluated using the Agilent 2200 TapeStation (Agilent Technologies), and each sample had the RNA Integrity Number (RIN) above 7.0. The ribosomal RNAs (rRNAs) were removed using Ribo-Zero rRNA Removal Kits (Illumina). RNA libraries were then constructed by NEBNext Ultra RNA Library Prep Kit for Illumina (New England Biolabs) and evaluated using the Agilent 2200 TapeStation and Qubit® 2.0 (Life Technologies). Sequencing was performed by RiboBio Co., Ltd. on an Illumina HiSeq 3000 machine with paired-end 150-bp reads. The adaptors and low quality bases assessed using FASTQC 0.11.3 were trimmed by Trimmomatic 0.39 using the following options: TRAILING:20, MINLEN:235 and CROP:235. Trimmed reads were then aligned to the ensembl 79 (GRCh38.p2) reference genome using STAR 2.4.2a. FeatureCounts 1.6.2 was subsequently employed to convert aligned short reads into read counts for each sample. Genes with less than ten counts in two or more samples were removed. The data were then analyzed using R 3.4.4 and DEseq2 1.18.1. Differentially expressed genes of each disease group were identified using Wald statistics test, with fold-change > 2 and Benjamini–Hochberg (BH)-adjusted P < 0.1 as compared to NC group. Results: These findings provide novel pathogenic links and molecular markers for better understanding and differential diagnosis of pulmonary TB, LUAD and sarcoidosis.

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RNAseq_HGSOC_marker_paper - samples

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