Project description:Purpose: The aim of this study was to determine the DNA methylation state of wildtype female mouse embryonic fibroblasts with nonsilencing shRNA mediated knockdown and Setdb1 geneTrap heterozygous cells with Setdb1 shRNA mediated knockdown. Methods: Enhanced Reduced Representation Bisulfite Libraries (eRRBS) were produced as previously descirbed (Akalin et al. 2012). Libraries were pooled and sequenced on the Illumina HiSeq 2000 platform for 100 bp single-end reads with dark cycle parameters (Boyle et al. 2012). Image analysis was performed in real time by the HiSeq Control Software (HCS) v1.4.8 and Real Time Analysis (RTA) v1.12.4.2, running on the instrument computer. Real-time base calling on the HiSeq instrument computer was performed with the RTA software. Illumina CASAVA1.8 pipeline was used to generate the sequence data.
Project description:Nipple aspriate fluid (NAF) were obtained from 7 women including 3 breast cancer patients. Cell-free DNA (cfDNA) were isolated and bisulfite sequencing using Illumina HiSeq X Ten platform.
Project description:As part of our study in understanding the role of SP140 in inflammatory pathways in macrophages, we inhibited SP140 mRNA using siRNA. Peripheral blood mononuclear cells (PBMCs) were obtained from whole blood of healthy donors (from Sanquin Institute Amsterdam or from GSK Stevenage Blood Donation Unit) by Ficoll density gradient (Invitrogen). CD14+ monocytes were positively selected from PBMCs using CD14 Microbeads according to the manufacturer’s instructions (Miltenyi Biotec). CD14+ cells were differentiated with 20 ng/mL of macrophage colony-stimulating factor (M-CSF) (R&D systems) for 3 days followed by 3 days of polarization into classically activated (inflammatory) M1 macrophages (100 ng/mL IFN-γ; R&D systems). M1 macrophages were transfected with siGENOME human smartpool SP140 siRNA or non-targeting scrambled siRNA for 48h with DharmaFECT™ transfection reagents according to manufacturer’s protocol (Dharmacon). The cells were left unstimulated or stimulated with 100 ng/mL LPS (E. coli 0111:B4; Sigma) for 4h (for qPCR) or 24h (for Elisa). The cells were lysed (ISOLATE II RNA Lysis Buffer RLY-Bioline) for RNA extraction.150 ng total RNA was labelled using the cRNA labelling kit for Illumina BeadArrays (Ambion) and hybridized with Ref8v3 BeadArrays (Illumina). Arrays were scanned on a BeadArray 500GX scanner and data were normalized using quantile normalization with background subtraction (GenomeStudio software; Illumina). This submission only contains processed data
Project description:To understand regulation mechanism of miR163, transcriptomes of WT and mir163 mutant plants were dissected. Total RNAs from 7-day-old seedlings were isolated for library construction and analyzed by RNA-seq via Illumina HiSeq platform. Raw sequences were obtained from the Illumina Pipeline software Illumina bcl2fastq v2.17.1.14 and expected to generate 6 G per sample.
Project description:Purpose: The aim of this study was to determine the DNA methylation state of wildtype female mouse embryonic fibroblasts with nonsilencing shRNA mediated knockdown and Setdb1 geneTrap heterozygous cells with Setdb1 shRNA mediated knockdown. Methods: Enhanced Reduced Representation Bisulfite Libraries (eRRBS) were produced as previously descirbed (Akalin et al. 2012). Libraries were pooled and sequenced on the Illumina HiSeq 2000 platform for 100 bp single-end reads with dark cycle parameters (Boyle et al. 2012). Image analysis was performed in real time by the HiSeq Control Software (HCS) v1.4.8 and Real Time Analysis (RTA) v1.12.4.2, running on the instrument computer. Real-time base calling on the HiSeq instrument computer was performed with the RTA software. Illumina CASAVA1.8 pipeline was used to generate the sequence data. Determine the DNA methylation state of mouse embryonic fibroblasts (MEFs) with wildtype MEFs and nonsilencing shRNA mediated knockdown or Setdb1 geneTrap heterozygous MEFs with Setdb1 shRNA mediated knockdown. Single-end with dark cycle protocol (Boyle et al. 2012)
