Transcriptome 3' end profiling by high throughput sequencing of HeLa cells depleted of MATR3, PTBP1/2 individually or in combination.
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ABSTRACT: We performed mRNA 3'end sequencing experiments on three biological replicates of HeLa cells depleted of MATR3, PTBP1/2, controls, or combined depletion of MATR3/PTBP1/2. Cells were fractionated into cytoplasmic and nuclear RNAn and only the nuclear RNA was used. Library preparation was done with the QuantSeq library kit (Lexogen) according to manufacturer’s recommendations. Replicates 1 and 2 were prepared with the QuantSeq forward library kit, replicates 3 and 4 with the QuantSeq reverse library kit. All libraries were sequenced on Illumina HiSeq2 machines in a single-end manner with a read length of 100 nt.
Project description:We performed RNA-seq experiments on three biological replicates of HeLa cells depleted of MATR3, PTBP1/2, controls, or combined depletion of MATR3/PTBP1/2. Cells were fractionated into cytoplasmic and nuclear RNA. Library preparation was done with the TruSeq stranded RNAseq library kit (Illumina) according to manufacturer’s recommendations; RNA was depleted of rRNA using the RiboZero kit (Epicentre). All libraries were sequenced on Illumina HiSeq2 machines in a single-end manner with a read length of 100 nt.
Project description:The aim of the experiment was to compare binding of PTBP1 in presence and absence of MATR3. HEK293T cells were transfected with siRNA targeting MATR3 or control sequences and labelled with 4thio-uridine for 8 hours (100µM, crosslinking 2x400mJ/cm2 365nm UV light).
Project description:The aim of the experiment was to compare binding of MATR3 in presence and absence of PTBP1. HEK293T cells were transfected with siRNA targeting PTBP1 and PTBP2 or control sequences and labelled with 4thio-uridine for 8 hours (100µM, crosslinking 2x400mJ/cm2 365nm UV light).
Project description:Neisseria meningitidis and Borrelia bavariensis are the causative agents of invasive meningococcal disease and Lyme neuroborrelosis. Before infecting the brain parenchyma the bacterial pathogens should cross the human blood-brain barrier (BBB). To understand the response of BBB against the invading pathogens, in vitro BBB spheroid model was generated by co cultivating human brain microvascular endothelial cells, pericytes and astrocytes in low adhesion conditions. Spheroids (N= 36) were infected with either N. meningitidis (6 x 10e4, MOI 1:4) or B bavariensis(1.5 x 10e5, MOI 1:10) for 3h. Two separate control groups of spheroids (N=36) without any infection were included in the study. After 3h of treatment (infection/no infection), 12 spheroids were pooled to form one biological sample and such 3 biological replicates per treatment were sampled for total RNA extraction. Thereafter, 12 cDNA libraries were prepared using QuantSeq FWD 3’mRNA Library Prep Kit (Lexogen). Second strand of cDNA library was synthesized using the UMI Second Strand Synthesis Mix (USS, Lexogen) containing 6 nucleotides long Unique Molecular Identifiers (UMIs). The double stranded cDNA libraries were amplified in 17-19 PCR cycles using i5 Unique Dual Indexing Add-on Kit (Lexogen). Illumina NextSeq 500 was employed to sequence the libraries having a read lengths of single-end 75 bp producing about 10 million reads per library. To carryout gene expression analysis, Bioconductor R-package - DESeq2 v1.20.0 (Love et al., 2014) was used where in, featureCounts tool v1.6.3 (Liao et al., 2013) was used to enumerate the genes. A minimum threshold of adjusted p-value < 0.05 and log2fold-change (log2FC) = ± 1 was set to considered the gene as differentially expressed (DEG).
Project description:3' mRNA-seq was performed with QuantSeq 3' mRNA-Seq kit (Lexogen 015) according to the manufacturer’s recommendations. 3' mRNA-seq was done in biological triplicates (soma) or duplicates (neurites), using 260 ng of total RNA from neurites or soma of mESC-derived neurons per sample. Libraries were pooled and sequenced on Illumina NextSeq 500 system with a single-end 150-cycle run.
Project description:The main aim was to determine how polyadenylation site usage is affected by osteoarthritis. Age matched tissue samples were obtained from both healthy individuals and those with late stage osteoarthritis, total RNA was isolated and then QuantSeq Reverse library produced using commercial kit (Lexogen) before sequencing
Project description:We describe an improved individual nucleotide resolution CLIP protocol (iiCLIP), which can be completed within 4 days from UV crosslinking to libraries for sequencing. For benchmarking, we directly compared PTBP1 iiCLIP libraries with the iCLIP2 protocol produced under standardised conditions with 1 million HEK293 cells, and with public eCLIP and iCLIP PTBP1 data. There are 3 PTBP1 iiCLIP libraries, 1 input iiCLIP library and 1 PTBP1 iCLIP2 library produced in this study.