Dataset Information

Ena-DATASET-TAU-03-04-2017-19:28:40:078-460 - samples

ABSTRACT: Targeted resequencing of samples was done with TruSeq custom amplicon low input kit (TSCA-LI, Illumina). The oligo capture probes were designed to include a prefix of 8 random nucleotides at the 5 end of each probe. The assay is designed such that each targeted locus is annealed with two probes, resulting in amplicons tagged with unique molecular identifiers (UMI) (22) of 16 bases. Raw FASTQ sequencing files were processed as following: (a) The first 8 bases were trimmed from each read and recorded with the corresponding base quality scores (BQ) in the attribute field. (b) Reads were aligned with BWA. (c) First round of PCR duplicate cleaning was performed with picard tools markDuplicates using the parameters BARCODE_TAG=BC TAGGING_POLICY=All REMOVE_DUPLICATES=true (d) Since in the previous step only duplicate reads with identical UMIs were removed, a second pass of filtering was done. Reads with identical mapping were considered unique only if their corresponding UMIs were different in at least 3 positions (i.e., UMI edit distance > 2). (e) Paired-end read pairs overlapping genomic positions were clipped to avoid overestimation of the sequencing coverage using bamUtils clipOverlap.

PROVIDER: EGAD00001003275 | EGA |

REPOSITORIES: EGA

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Publications

Suppressors and activators of JAK-STAT signaling at diagnosis and relapse of acute lymphoblastic leukemia in Down syndrome.

Schwartzman Omer O Savino Angela Maria AM Gombert Michael M Palmi Chiara C Cario Gunnar G Schrappe Martin M Eckert Cornelia C von Stackelberg Arend A Huang Jin-Yan JY Hameiri-Grossman Michal M Avigad Smadar S Te Kronnie Geertruy G Te Kronnie Geertruy G Geron Ifat I Birger Yehudit Y Rein Avigail A Zarfati Giulia G Fischer Ute U Mukamel Zohar Z Stanulla Martin M Biondi Andrea A Cazzaniga Giovanni G Vetere Amedeo A Wagner Bridget K BK Chen Zhu Z Chen Sai-Juan SJ Tanay Amos A Borkhardt Arndt A Izraeli Shai S

Proceedings of the National Academy of Sciences of the United States of America 20170501 20

Children with Down syndrome (DS) are prone to development of high-risk B-cell precursor ALL (DS-ALL), which differs genetically from most sporadic pediatric ALLs. Increased expression of cytokine receptor-like factor 2 (CRLF2), the receptor to thymic stromal lymphopoietin (TSLP), characterizes about half of DS-ALLs and also a subgroup of sporadic "Philadelphia-like" ALLs. To understand the pathogenesis of relapsed DS-ALL, we performed integrative genomic analysis of 25 matched diagnosis-remissio ...[more]

PMID: 28461505

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Project description:Purpose: To analyze the sensitivity and specificity of the AmpFISH method, we sequenced the NIH3T3 cell line via UMI-RNAseq experiments. Methods:NIH3T3 cells were grown in DMEM (Dulbecco’s Modified Eagle Medium, Gibco) supplemented with 10% FBS (Fetal Bovine Serum, Sigma), 50U/ml Penicillin and 50 mg/ml streptomycin (Gibco,cat.no.15070) at 37℃ with 5% CO2. Cells were treated with 0.25% trypsin solution (HyClone, No.SH42605.01) when they reached ~106 cells/ml. Then, the cells were washed with 1X PBS, and then mixed with 1ml TRIzol solution (ThermoFish, No.15596029), and snap-frozen with dry ice. Total RNA was qualitatively and quantitatively evaluated as follows: (1) the RNA sample was initially qualitatively evaluated using 1% agarose gel electrophoresis for possible contamination and degradation; (2) RNA purity and concentration were then examined using NanoPhotometer spectrophotometer; (3) RNA integrity and quantity were finally measured using RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system. After library preparation and pooling of different samples, the samples were subjected to Illumina sequencing. The libraries were sequenced using the Illumina NovaSeq 6000 Platform for 6G raw data and generated 150nt pair-end reads. UMI sequences on each read were identified by UMI-tools (1.0.0), and reads with UMIs were used for the subsequent analysis. To identify the duplicated reads, UMIs were initially removed from the UMI reads, and the remaining parts of each read were mapped to the reference genome using Hisat2. Reads that mapped to the same location on the reference genome were identified as duplicated reads. Then, the UMIs on each read were recalled, and the duplicated reads with the same UMI were identified as non-natural duplications, which were subsequently removed from the processed data. HTSeq v0.6.1 was used to count the read numbers mapped to each gene. Then, the FPKM of each gene was calculated based on the length of the gene, and the read count was mapped to the gene. Conclusions:AmpFISH provides convenient and versatile tools for sensitive RNA/DNA detection and to gain useful information on cellular molecules using simple workflows

Project description:A set of 5,908 69-70 mers oligonucleotides that correspond to all C. glabrata putative open reading frames and some non-coding sequences was designed and synthesized at Pasteur Institute, France. Each oilgonucleotide was dissolved into 50% DMSO and spotted on UltraGAPsTM glass slides (Corning Incorporated, Acton, MA) in duplicate to generate whole genome microarray for C. glabrata. The microarray was printed at Biotechnology Research Institute/National Research Council of Canada in Montreal. The sequence information of the oligonucleotides and the micorarray setup can be found at platform GPL3922. Using the microarray, the transcriptional profiles of C. glabrata cells that were coincubated with J774A.1 murine Macrophage-like cells for 2 and 6 hrs were examined. At each time point, three biological repeats were done. 20 ug of total RNA from either macrophage-ingested C. glabrata cells (experimental sample) or the medium-alone controls (control sample) was used to synthesize Cy5- or Cy3-labeled cDNA probes with an anchor primer [(dT)18(dA/dG/dC)] in the presence of Cy5- or Cy3-dCTP (PerkinElmer). For each pair of experimental and control samples, dye-swap was performed, that is, two probe mixtures, the Cy5-experimental plus the Cy3-control probes, and conversely, the Cy3-experimental plus the Cy5-control probes were individually hybridized with a single C. glabrata microarray slide. The hybridization and subsequent washing conditions follows the instruction on the manual for UltraGAPsTM glass slides. The hybridized microarrays were scanned with Axon 4000B scanner, and Data were extracted using Axon GenePix program. Log2 (experimental/control) for each spot on the microarray was calculated. From each dye-swap experiment, the Log2 (experimental / control) for each spot were averaged. Finally, 6 Log2 (experimental/control) values (3 repeats × duplicate spots on each slide) were imported into SAM (significance analysis of microarrays) software for statistical analysis. For genes that are considered significantly induced or repressed, the median of false discovery rate (FDR) equals to 0, while the 90% FDR is less than 0.001. Keywords: transcriptional profiling by microarray

Dataset Information

Ena-DATASET-TAU-03-04-2017-19:28:40:078-460 - samples

Publications

Suppressors and activators of JAK-STAT signaling at diagnosis and relapse of acute lymphoblastic leukemia in Down syndrome.

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