Project description:Whole genome sequencing of 10 HCLc tumor and matched-germline T cells. Genomic DNA from highly purified HCLc tumor and T cell populations were utilized for library preparation using NEBNext Ultra DNA library prep kit. Sequencing was performed as 150 bp paired end sequencing using four lanes of an Illumina HiSeq4000 to an average depth of 12X. Reads from each library were aligned to the human reference genome GRCh37 using BWA-MEM (v0.7.12). The analysis of somatic genetic alterations in WGS data from tumor-germline pair HCLc samples was divided based on the nature of the mutation, as follow: single-nucleotide variants (SNVs), indels, CNAs and SVs. Moreover, COSMIC mutational signatures and subclonal architecture was inferred for each tumor.
Project description:Urine from a patient with a urinary tract infection was plated on LB agar plate. Microcolonies appeared ~8h after plating. Microcolonies were picked and subjected to Microcolony-seq and to whole-genome sequencing. Genomic DNA of each UTI microcolony was extracted from 1 mL of bacteria using the DNeasy Blood & Tissue Kit (Qiagen). Library preparation and sequencing was carried out by BGI company, China. Concentration of samples was detected by fluorometer or Microplate Reader (Qubit Fluorometer, Invitrogen). Sample integrity and purity were detected by Agarose Gel Electrophoresis. 1μg genomic DNA was randomly fragmented by Covaris. The fragmented genomic DNA were selected by Agencourt AMPure XP-Medium kit to an average size of 200-400bp. Fragments were end repaired and then 3’ adenylated. Adaptors were ligated to the ends of these 3’ adenylated fragments. PCR products were purified by the Agencourt AMPure XP-Medium kit. The double stranded PCR products were heat denatured and circularized by the splint oligo sequence. The single strand circle DNA (ssCir DNA) were formatted as the final library. Samples were deep-sequenced with the DNBseq G400 machine using the 150-cycles paired-end with 350 bp insert size. At least 150X sequencing depth for each nucleotide in each sample was targeted.
Project description:Purpose: The study aimed to characterize the molecular phenotype of bone marrow macrophages in NHL with RNA sequencing analysis. Methods:RNA of sorted femur macrophages (CD11b+F4/80+) were extracted with an RNA extraction kit (Qiagen). The samples were submitted to Novogene Inc. for library preparation and subsequent RNA sequencing. RNA was used for cDNA library construction using an NEBNext® Ultra RNA Library Prep Kit for Illumina® (New England Biolabs) according to the manufacturer’s protocol. The resulting 250-350 bp insert libraries were quantified using a Qubit 2.0 fluorometer (Thermo Fisher Scientific) and quantitative PCR. Size distribution was analyzed using an Agilent 2100 Bioanalyzer (Agilent Technologies). Qualified libraries were sequenced on an Illumina HiSeq 4000 Platform (Illumina) using a paired-end 150 run (2×150 bases). Reads containing adapter or poly-N and those of low quality were trimmed, after which gene counts were obtained though mapping the clean reads to reference genome mm10 using STAR 2.5.3a.
Project description:Purpose: Examination of different forms of tocopherol treatment in mammary tumorigenesis by comparing NGS-derived transcriptome profiling (RNA-seq) Methods: Use of cDNA library construction and Illumina sequencing (NextGen 75 bp pair-end) and quality control using FastQC. Results: Differential transcriptome profiling of mammary tumors between different forms of tocopherol treatment was observed. Conclusions: Our study represents the first detailed analysis of mammary tumor transcriptomes with different tocopherol treatment generated by RNA-seq technology. The optimized data analysis reported here should provide a framework for comparative investigations of expression profiles with tocopherols.
Project description:The intent of the experiment was to infer, from re-sequencing of genomic DNA, the coordinates of neo-insertions from activated ONSEN/COPIA78 LTR retrotrasnposon in Arabidopsis thaliana. For this, we performed Illumina 75 bp pair-end PCR-free DNA genome re-sequencing in several independent lines of RdDM mutant nrpd1-3.
Project description:The intent of the experiment was to infer from DNA sequencing the occurrence of extra-chromosomal DNA from Arabidopsis thaliana's heat-activated LTR retrotransposon Onsen/COPIA78. For this, we performed Illumina 150 bp pair-end PCR-free DNA genome re-sequencing, in both wild-type Col-0 and RdDM mutant nrpd1-3 under control and heat stress.
Project description:RNA Sequencing analysis was performed on RNA isolated from tissues(forebrain,midbrain and hindbrain) from P-25 wild type and Fmr1CRE knockin mutant mice from same litter.Random primed cDNA from poly(A) selected RNA was converted into an Illumina sequencing library using RNA Library Prep Kit from Illumina (E7420, NEB, USA) in conjunction with NEBNext® Multiplex Oligos for Illumina (E7335/E7500, NEB, USA). and single-end 50-base pair (bp) reads were generated using a NextSeq 500 (Illumina Inc, SY-415-1002). Eighteen libraries were combined in two equimolar pools of 9 based on the library quantification results and each pool was run across a single High-Output Flow Cell. Sequencing was performed at the Wellcome Trust Clinical Research Facility (WTCRF; Edinburgh). Fastq files were processed to transcript-level counts and quality control performed using the bcbio_nextgen pipeline and the illumina-RNAseq workflow template. Differential Expression (DE) analysis was performed in R. The R package bcbio-RNAseq was used to import salmon transcript level counts into DESeq2.
Project description:To directly test how individual TFs combine their functions to open chromatin at specific loci, we used synthetic DNA sequences. We generated a library containing all combinations of TF-motif deletions for three CREs: a CTCF-bound region as a positive control (found in \"MutLib1\"), an enhancer with five KLF4 motifs (found in \"MutLib2\"), and a promoter with YY1, NRF1, and MYC motifs (found in \"MutLib1\"). We then inserted the library into a landing pad within a neutral chromatin environment, devoid of activating or repressive chromatin modifications, in mESCs using Recombination-Mediated Cassette Exchange (RMCE). Chromatin accessibility of the inserted fragments was then profiled by targeted SMF using PCR primers that anneal to a synthetic flanking sequence, allowing unambiguous differentiation of the ectopic site from its endogenous counterpart. The dataset generated includes amplicon-based SMF data from the ectopic site under steady-state conditions, in mouse cells (i.e., TC-1 knock-out of the three DNA methyl transferases (DNMT TKO) mESCs). Two biological replicates were generated. In summary, cells were collected for SMF, which marks accessible cytosines via recombinant methyltransferases, followed by bisulfite sequencing to infer protein-DNA interactions and chromatin accessibility at single-molecule resolution. The sequencing library was prepared using the NEBNext DNA Ultra II Library Prep Kit and sequenced on an Illumina platform, using either a MiSeq 250 bp paired-end run, a MiSeq i100 250 bp paired-end run, or a NextSeq 2000 P1 300 bp paired-end run. Reads were pre-processed with TrimGalore and a custom R script was used to trim the plasmid backbone from the reads. After, pre-processed reads were aligned using QuasR. Further analyses were conducted using custom scripts available at https://github.com/Krebslabrep/TF-chromatin.git.
Project description:The DNA methylome of 45 primary neuroblastoma tumors is profiled by enrichment with a methyl-CpG-binding domain (MBD) and massively parallel sequencing DNA of 45 primary tumors is sheared (fragments of ± 200 bp), followed by MBD-based (MethylCap kit of Diagenode) enrichement, library preparation and multiplexing. Both input DNA and captured DNA were sequenced paired-end on Illumina Hiseq2000
Project description:8 neuroblastoma (NB) cell lines (CLB-GA, IMR-32, SH-SY5Y, N206, CHP-902R, LAN-2, SK-N-AS, SJNB-1) their methylome is determined by sequencing after MBD2-capture using MethylCollector (ActiveMotif) 8 NB cell lines were included (CLB-GA, IMR-32, SH-SY5Y, N206, CHP-902R, LAN-2, SK-N-AS, SJNB-1) in this study. After shearing (fragments of about 200 bp), DNA was captured using MBD2-capture (MethylCollector - ActiveMotif) followed by library preparation and multiplexing. Captured sequence tags were sequenced paired-end (2 x 45 bp) on Illumina GAIIx.