IiCLIP protocol benchmarking with PTBP1
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Illumina MiSeq, Illumina HiSeq 4000
ORGANISM(S): Homo sapiens
SUBMITTER: Flora Lee
PROVIDER: E-MTAB-10881 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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2018-06-15 | E-MTAB-6927 | biostudies-arrayexpress
Project description:PTBP1 and PTBP2 control alternative splicing programs during neuronal development, but the cellular functions of most PTBP1/2-regulated isoforms remain unknown. We show that PTBP1 guides developmental gene expression by regulating the transcription factor Pbx1. We identify exons that are differentially spliced when mouse embryonic stem cells (ESCs) differentiate into neuronal progenitor cells (NPCs) and neurons, and transition from PTBP1 to PTBP2 expression. We define those exons controlled by PTBP1 in ESCs and NPCs by RNA-seq analysis after PTBP1 depletion and PTBP1 crosslinking-immunoprecipitation. We find that PTBP1 represses Pbx1 exon 7 and the expression of its neuronal isoform Pbx1a in ESC. Using CRISPR-Cas9 to delete regulatory elements for exon 7, we induce Pbx1a expression in ESCs, finding that this activates transcription of specific neuronal genes including known Pbx1 targets. Thus PTBP1 controls the activity of Pbx1 and suppresses its neuronal transcriptional program prior to differentiation. 46C mESCs were treated with 20 nM control, Ptbp1, Ptbp2, or Ptbp1 and Ptbp2 siRNAs for 72 hours. The knockdowns were performed using 2 independent sets of siRNAs, including one biological replicate. Poly-A RNA was isolated for RNA-sequencing and splicing analyses.
2016-07-03 | E-GEOD-71075 | biostudies-arrayexpress
Project description:Background: Genome-wide transcriptome profiling generated by microarray and RNA-Seq often provides deregulated genes or pathways applicable only to larger cohort. On the other hand, individualized interpretation of transcriptomes is increasely pursued to improve diagnosis, prognosis, and patient treatment processes. Yet, robust and accurate methods based on a single paired-sample remain an unmet challenge. Methods: M-bM-^@M-^\N-of-1-pathwaysM-bM-^@M-^] translates gene expression data profiles into mechanism-level profiles on single pairs of samples (one p-value per geneset). It relies on three principles: i) statistical universe is a single paired sample, which serves as its own control; ii) statistics can be derived from multiple gene expression measures that share common biological mechanisms assimilated to genesets; iii) semantic similarity metric takes into account inter-mechanismsM-bM-^@M-^Y relationships to better assess commonality and differences, within and cross study-samples (e.g. patients, cell-lines, tissues, etc.), which helps the interpretation of the underpinning biology. Results: In the context of underpowered experiments, N-of-1-pathways predictions perform better or comparable to those of GSEA and Differentially Expressed Genes enrichment (DEG enrichment), within- and cross-datasets. N-of-1-pathways uncovered concordant PTBP1-dependent mechanisms across datasets (Odds-RatiosM-bM-^IM-%13, p-valuesM-bM-^IM-$1M-CM-^W10-5), such as RNA splicing and cell cycle. In addition, it unveils tissue-specific mechanisms of alternatively transcribed PTBP1-dependent genesets. Furthermore, we demonstrate that GSEA and DEG Enrichment preclude accurate analysis on single paired samples. Conclusions: N-of-1-pathways enables robust and biologically relevant mechanism-level classifiers with small cohorts and one single paired samples that surpasses conventional methods. Further, it identifies unique sample/ patient mechanisms, a requirement for precision medicine. Cell lines, culture conditions: The epithelial human breast cancer cell line MDA-MB231 (ER-/ PR-/ HER2-) were obtained from the American Type Culture Collection (Manassas, VA). The epithelial human ovarian tumor cell line A2780 was received as a generous gift from Dr. Thomas C. Hamilton (Fox Chase Cancer Center, Philadelphia, PA) Cancer Center, Philadelphia, PA). Cells were grown in DMEM supplemented with 10% fetal bovine serum (FBS), 2mM L-glutamine in a humid environment at 37M-BM-0C, with 5% CO2. Both cell lines were free of Mycoplasma species and were maintained for no longer than 10 weeks in culture after recovery from frozen stocks. Mycoplasma levels were checked periodically using the MycoAlertM-BM-. Mycoplasma Detection Kit (Lonza Inc., Allendale, NJ). The authenticity of cell lines was assessed by the ATCC carrying out short tandem repeat (STR) analysis (Verified STR Profiling Service, ATCCM-BM-. 135-XV). Additionally, we compared A2780 to the original STR profile collected by the European Collection of Cell Culture (Catalogue number 93112519). Doxycycline-inducible knockdown of PTBP1 regulated by small hairpin RNA (shRNA): In order to analyze the effect of PTBP1 depletion, two consecutive viral transductions were performed in both MDA-MB231 and A2780 cell lines. Cells were plated on 24-well plate (10-20M-CM-^W104 cells/well), maintained in culture for 16 hours, and then medium containing LV-tTR/KRAB-Red lentiviral particles was added. Following 16 h of incubation, cells were transduced a second time by LV-THM/PTBshRNA or LV-THM/LUCshRNA lentiviral particles. Clones expressing both red and green fluorescent protein (dsRED and GFP respectively) were selected and expanded. Following 16 h of incubation, cells were washed and split in two subcultures, one without doxycycline (PTBP1/-DOX; Control in Figure 1) and the other with Doxycycline (DOX) at a final concentration of 1 M-NM-<g/ml (PTBP1/+DOX; PTBP1-KD in Figure 1). Doxycycline was prepared according to the manufacturerM-bM-^@M-^Ys recommendations (Sigma-Aldrich, St. Louis, MO). Five days later, cells were analyzed by fluorescence microscopy, and PTBP1 gene expression was assessed using PCR and Western Blotting (data not shown). The cells that were transduced by LV-LUCshRNA express PTBP1 regardless of the presence of DOX (LUCshRNA/+DOX). Constructs and lentivirus preparation were performed as previously described in literature [He X et al., Knockdown of polypyrimidine tract-binding protein suppresses ovarian tumor cell growth and invasiveness in vitro, Oncogene 2007, 26(34):4961-4968]. Microarray Analysis: For each of the cell lines, MDA-MB231 and A2780, total RNAs were extracted from four biological replicates of PTBP1-depleted cells, PTBP1-KD (4M-CM-^W PTBP1/+DOX) and eight biological replicates control cells (4M-CM-^W PTBP1/-DOX and 4M-CM-^W LUCshRNA/+DOX) by Direct-zol RNA kit (Zymo Research, Irvine, CA). All paired samples consist of PTBP1-depleted cells (PTBP1-KD) and matched control cells. Qualities of RNA were assessed based on the RNA quality indicator (RQI M-bM-^IM-% 8) using Experion Automated Electrophoresis System (Bio-Rad, Hercules, CA). Gene expression microarray measurements were performed using the GeneChip PrimeView Human Gene Expression Array that contains 49,395 probes and measures 36,000 transcripts and variants per sample. Labeling and hybridization were performed following Affymetrix protocols. The raw data were normalized according to the Robust Multiple-array Average (RMA) technique, using Affymetrix Power Tools (APT). biological replicate: Ovarian_LucsH_DOX_1, Ovarian_LucsH_DOX_2, Ovarian_LucsH_DOX_3, Ovarian_LucsH_DOX_4 biological replicate: Ovarian_PTBsh1_NODOX_1, Ovarian_PTBsh1_NODOX_2, Ovarian_PTBsh1_NODOX_3, Ovarian_PTBsh1_NODOX_4 biological replicate: Ovarian_PTBsh1_DOX_1, Ovarian_PTBsh1_DOX_2, Ovarian_PTBsh1_DOX_3, Ovarian_PTBsh1_DOX_4 biological replicate: Breast_LucsH_DOX_1, Breast_LucsH_DOX_2, Breast_LucsH_DOX_3, Breast_LucsH_DOX_4 biological replicate: Breast_PTBsh1_NODOX_1, Breast_PTBsh1_NODOX_2, Breast_PTBsh1_NODOX_3, Breast_PTBsh1_NODOX_4 biological replicate: Breast_PTBsh1_DOX_1, Breast_PTBsh1_DOX_2, Breast_PTBsh1_DOX_3, Breast_PTBsh1_DOX_4
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