3prime RNA-seq of murine SCLC cell line modified with CRISPR-activation of Myc, Mycl or Mycn
ABSTRACT: In cells derived from a genetically engineered Rb1 and Trp53 loss mouse model of SCLC (RP) expression of one of the three MYC family members induced from the endogenous locus using CRISPR activation. Cells were first stably transfected with the lenti-MS2-p65-HSF1 activator plasmid. Respective sgRNAs targeting either Myc, Mycl or MYCN were then cloned into the lentiSAMv2 system and transfected separately into the MS2-p65-HSF1 cells using lentiviral delivery.
Project description: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:Differential gene expression profiling was performed in two lymphoblastoid cell lines with different radiosentivitity, one radiosensitive (RS) and another radioresistant (RR), after different post-irradiation times. A greater and a prolonged transcriptional response after irradiation was induced in the RS cell line. Functional analysis showed that 24 h after irradiation genes involved in DNA damage response, negative regulation of the cell cycle and apoptosis were still differentially up-regulated in the RS cell line but not in the RR cell line. Sham-irradiated and irradiated (2 Gy) cell cultures of the RS and the RR cell line were incubated at 37ºC for 4 and 24 h and 14 days. After that, RNA was extracted and sequenced with QuantSeq technology
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:Gene analysis of HCC cells transfected with Lenti-vector, Lenti-MAOA and stimulated with Norepinephrine Total mRNA obtained from SMMC-7721 cells transfected with Lenti-vector, Lenti-MAOA and stimulated with Norepinephrine
Project description:We purified FAPs from the hind limbs of wild type, mdx and cardiotoxin-injured mice in order to unveil changes in their transcriptomes. RNA was isolated directly from sorted cells and analyzed by 3’ RNA sequencing.
Project description:HSF1 orchestrates the heat shock response pathway. This pathway is co-opted in cancer and provides critical stress relief from oncogenic stress. HSF1 has a diverse occupancy signature depending on the cell type. In this study, we performed HSF1 ChIP-seq analysis using the human T-ALL cell line CUTLL1 and P12. These results revealed the HSF1 chromatin binding signature in CUTLL1 and P12 cells. MYC is a driving oncogene in T-ALL. The non-oncogene addiction pathways that act downstream of this transcription factor to support anabolic pathways are not well-understood. For this reason, we performed MYC ChIP-seq analysis using the human T-ALL cell line CUTLL1. These results revealed that HSF1 and HSF1 targets are included in the MYC binding signature. Overall design: Twenty million cells were used for the ChIP and precipitated using 5 micrograms of antibody (cell signaling, 4356) against human HSF1. Twenty million cells were used for the ChIP and precipitated using 5 micrograms of antibody (santa cruz biotechnology, N-262) against human MYC
Project description:The mRNA m6A reader YTHDF2 is overexpressed in a broad spectrum of human acute myeloid leukemias (AML). To study the role of YTHDF2 on mRNA decay rates in leukemia, c-Kit+ cells from foetal livers of Ythdf2fl/fl; Vav-iCre (Ythdf2CKO) and Ythdf2fl/fl (Ythdf2CTL) 14.5 dpc embryos were transduced with Meis1 and Hoxa9 oncogenes and serially re-plated to generate pre-leukemic cells. Medium with 4SU was used for pre-leukemic cells labelling for 12 hours and was later replaced with 4SU-free medium (time 0). Cells were collected immediately after medium change and at 1, 3 and 9 hours for library generation. RNA from Ythdf2CKO (n=3 biological replicates) and Ythdf2CTL (n=3 biological replicates) pre-leukemic cells were used for SLAM-seq library generation.