EGAD00001003460
None
Illumina HiSeq 2000
Illumina HiSeq 2500
13
EGAD00001003808
None
Illumina HiSeq 2500
47
EGAD00001004810
This dataset is related to publications Costa et al. Cancer Cell 2018 and Givel et al. Nat. Commun. 2018 which describe the identification of 4 Cancer Associated Fibroblasts (CAF) in breast and ovarian cancer. This dataset contains transcriptomic profiles obtained by RNA-Seq of 34 CAF-S3 samples from breast and ovarian Tumors.
Illumina HiSeq 2500
34
EGAD00001005744
None
Illumina HiSeq 2500
28
EGAD00001006144
None
Illumina NovaSeq 6000
7
EGAD00001006145
None
Illumina NovaSeq 6000
70
EGAD00001006163
None
Illumina HiSeq 2500
Illumina NovaSeq 6000
8
EGAD50000000135
Bulk RNA-seq
Illumina NovaSeq 6000
5
EGAD50000000136
Spatial Transcriptomic
Illumina NovaSeq 6000
2
EGAD50000000190
Spatial transcriptomic data of HGSOC patients before and after treatment
Illumina NovaSeq 6000
10
EGAD50000000191
scRNAseq data of HGSOC patients before and after treatment
Illumina NovaSeq 6000
12
EGAD50000000192
Bulk RNAseq of cultured fibroblasts
Illumina NovaSeq 6000
6
EGAD50000000320
Fastq files of bulk RNAseq data from DCIS, invasive and microinvasive breast cancer at diagnosis. This dataset covers 18 DCIS cases, 17 microinvasive and 20 primary invasive breast cancer.
Illumina HiSeq 2500
55
EGAD50000000321
Fastq files from scRNAseq data of cancer associated pericytes-like from 3 patients.
CAP were isolated from a total of 3 primary BC (surgical residues prior to any treatment) by using BDFACS ARIA III sorter (BD Biosciences). BC were collected directly from the operating room after surgical specimen macroscopic examination and selection of areas of interest by a pathologist. Samples were cut into small pieces (around 1 mm3) and digested in CO2-independent medium (Gibco #18045-054) supplemented with 150 μg/mL liberase (Roche #05401020001) and Dnase I (Roche #11284932001) for 40 min at 37°C with shaking (180 rpm). After digestion, cells were processed and stained as described above (#Flow Cytometry analysis of BC samples). CAP fibroblasts were then gated on the Live/Dead negative fraction and defined as EPCAM- CD45- CD31- CD235a- FAPMed CD29High.
CAP scRNA-seq: Upon isolation, CAP cells were directly collected into RNase-free tubes (Thermo Fisher Scientific, #AM12450) precoated with DMEM (GE Life Sciences, #SH30243.01) supplemented with 10% FBS (Biosera, #1003/500). Single-cell capture, lysis, and cDNA library construction were performed using Chromium system from 10X Genomics, with the following kits: Chromium Single Cell 3′ Library & Gel Bead Kit v2 kit (10X Genomics, #120237) and Chromium Single Cell A Chip Kits (10X Genomics, #1000009). Generation of gel beads in Emulsion (GEM), barcoding, post GEM-reverse transcription cleanup and cDNA amplification were performed according to the manufacturer’s instructions. Cells were loaded accordingly on the Chromium Single cell A chips, and 12 cycles were performed for cDNA amplification. cDNA quality and quantity were checked on Agilent 2100 Bioanalyzer using Agilent High Sensitivity DNA Kit (Agilent, #5067-4626) and library construction followed according to 10X Genomics protocol. Libraries were next run on the Illumina HiSeq (for patients P1) and NovaSeq (for patients P2–3) with a depth of sequencing of 50,000 reads per cell.
Illumina NovaSeq 6000
3
EGAD50000000322
Fastq files from spatial transcriptomic of breast cancer coming from 8 Breast cancer sections.
Sample preparation: frozen BC samples were chosen based on tissue structure and RNA quality (RIN > 8). The “Visium Spatial Tissue Optimization Slide and Reagent Kit” (10X Genomics; #PN-1000193) was then used to optimize permeabilization conditions for BC tissues. Briefly, sections were fixed, stained and then permeabilized at different time points to capture mRNA, and the reverse transcription was performed to generate fluorescently labeled cDNA. The permeabilization time that resulted in the highest fluorescence signal with the lowest background diffusion was chosen. The best permeabilization time for BC tissue was 18 min. Cryostat sections of 10 μm of thickness were cut and placed on Visium Spatial Gene Expression slides (10X Genomics, PN-1000184). The slide was incubated for 1 min at 37°C, then fixed with methanol for 30 min at -20°C followed by Hematoxylin and Eosin (H&E) staining and images were taken under a high-resolution microscope. After imaging, the coverslip was detached by holding the slide in water and the slide was mounted in a plastic slide cassette. The spatial gene expression process, including tissue permeabilization, second strand synthesis and cDNA amplification, was performed according to the manufacturer’s instructions (10X Genomics; #CG000239). cDNA quality was next assessed using Agilent High sensitivity DNA Kit (Agilent, #5067-4626). The spatial gene libraries were constructed using Visium Spatial Library Construction Kit (10X Genomics, PN-1000184).
Illumina NovaSeq 6000
8
EGAD50000000323
Fastq files from bulk RNAseq of fibroblasts after culture and facs sorting (N=9).
Sorted FAP+ CAF cells RNAs were extracted using Qiagen miRNeasy Kit (Qiagen, #217004) according to the manufacturer's instructions. Verification of RNA integrity and quality was performed using the Agilent RNA 6000 nano Kit (Agilent Technologies, #5067-1511). cDNA libraries were prepared using the TruSeq Stranded mRNA Kit (Illumina, #20020594) followed by sequencing on NovaSeq (Illumina).
Illumina NovaSeq 6000
9
EGAD50000001121
Fastq files of single-cell RNAseq data from from Cancer Associated Fibroblasts (CAFs), cancer cells and immune cells isolated from 6 primary Invasive Lobular Carcinomas (ILCs)
Illumina NovaSeq 6000
6
EGAD50000001296
8 Fastq files of single-cell RNAseq data from tumor-on-chip (TOC) after 42h of TNBC and CAF mono- or co-culture
Illumina NovaSeq 6000
4
EGAD50000002494
Fastq files from 4 Visium spatial transcriptomic ILC sections
Illumina NovaSeq 6000
4
EGAD50000002850
Sucrose density gradient centrifugation was performed to separate the subpolysomal and polysomal ribosome fractions. For polysome profiling 30x10^6 IGROV1 at 70% confluence were needed after treatment with 10 µM MAP3K8-ki or DMSO for 1 hour before 10% FBS stimulation for 2 h. For cytoplasmic lysate preparation, 15 minutes prior to collection, the cells were incubated at 37 °C with 100 µg/ml cycloheximide, then washed, scraped into ice-cold PBS supplemented with 100 mg/ml cycloheximide, centrifuged at 3,000 r.p.m. for 5 min and resuspended into 400 µl of LSB buffer (20 mM Tris, pH 7.4, 100 mM NaCl, 3 mM MgCl2, 0.5 M sucrose, 2.4% Triton X-100, 1 mM DTT, 100 U/ml RNasin and 100 µg/ml cycloheximide). After homogenization, 400 µl of LSB buffer supplemented with 0.2% Triton X-100 and 0.25 M sucrose were added. The samples were centrifuged at 12,000g for 10 min at 4 °C. The resulting supernatant was adjusted with 5 M NaCl and 1 M MgCl2. The lysates were loaded onto a 15–50% sucrose density gradient and centrifuged in an SW41 rotor at 38,000 r.p.m. for 2 h at 4 °C. The polysomal fractions were monitored and collected using a gradient fractionation system (Isco). Three independent experiments were performed, and total RNA was extracted from 75µl of the four heaviest fractions pooled and input samples using the TRIzolTM
-LS (ThermoFisher Scientific #10296010) according to the manufacturer’s instructions for polysome-bound mRNA-sequencing (polysome-bound mRNA-seq) and total mRNA-sequencing (mRNA-seq), respectively. RNA integrity and quality were analyzed with the Agilent RNA 6000 Pico kit. RNA sequencing libraries were prepared from total RNA prep using the Illumina® Stranded mRNA Prep Ligation library preparation kit, which allows to perform a strand specific sequencing. This protocol includes a first step of polyA selection using magnetic beads to focus sequencing on polyadenylated transcripts. After RNA fragmentation, cDNA synthesis was then performed and resulting fragments were used for dA-tailing followed by ligation of RNA Index Anchors. PCR amplification with indexed primers (IDT for Illumina RNA UD Indexes) was finally achieved, with 15 cycles, to generate the final cDNA libraries. Individual library quantification and quality assessment was performed using Qubit fluorometric assay (Invitrogen) with dsDNA HS (High Sensitivity) Assay Kit and LabChip GX Touch using a High Sensitivity DNA chip (Perkin Elmer).
Libraries were then equimolarly pooled and quantified by qPCR using the KAPA library quantification kit (Roche). Sequencing was carried out on the NovaSeq 6000 instrument from Illumina using paired-end 2 x 100 bp, to obtain around 30 million clusters (60 million raw paired-end reads) per sample.
Illumina NovaSeq 6000
28
EGAD50000002851
RPE cell lines were transfected with a GFP empty vector as a control, QARS wild-type (wt), and a catalytically inactive QARS mutant. To investigate the impact of QARS in its active versus inactive form on DNA damage repair factors, three treatment conditions were applied: (1) untreated, (2) pulse, where the DNA-damaging drug was administered for 1 hour, and (3) a washout condition, where the drug was applied for 1 hour and then removed, followed by a 5-hour recovery period to allow for DNA repair assessment.
Illumina NovaSeq X
25
