Supplementary MaterialsSupplementary material 1 (DOCX 34?kb) 18_2017_2580_MOESM1_ESM. to be highly expressed in differentiated neural stem cells across different time points of differentiation, and its expression correlated with gene expression. Concomitant expression of and was further observed in several cancer cell models. While the function of this transcript is unknown, the regulatory role reported for other lncRNAs strongly suggests a possible role for in regulating expression, simply because previously observed for appearance will donate to clarifying its function in stem cell tumorigenesis and differentiation. Electronic supplementary materials The online edition of the content (doi:10.1007/s00018-017-2580-3) contains supplementary materials, which is open to authorized users. gene maps to Chr3q26.3, in a intron of an extended non-coding RNA (LncRNA) called overlapping transcript (overlapping transcripts are reported to get multiple transcription begin sites (TSS) also to be transcribed into several substitute transcript variations [5]. Lately, concomitant gene appearance of and it has been reported in breasts, lung and oesophageal carcinoma [6C8]. Current research suggest a confident function for in regulating is certainly differentially spliced into multiple transcript variations in stem and tumor cells, and it has been suggested to are likely involved in regulating appearance of [9, 10]. Open up in another window Fig.?1 Structural similarity between SOX1 and SOX2 loci. a Snapshot pictures from the individual locus on individual chromosome 3 extracted from the UCSC genome browser showing the gene itself (non-coding gene within which lies. b Snapshots of the human locus on human chromosome 13 taken from the UCSC genome browser showing that similarly to SOX2, the gene is usually annotated within a larger non-coding gene (LINC00403, in a and b are the SOX2 and SOX1 genes, respectively locus has been studied far less than that of locus which was found to harbour an overlapping transcript, and describe expression, splicing variants and detection in different stem cell and cancer cell models. Materials and methods Reagents were purchased from ThermoFisher (UK) unless otherwise stated. Cell sample preparation Cells lines used in this study are described in Table?1. Ntera2, hMSCs, HeLa, SH-SY5Y and HOS cell lines were produced in Dulbecos Modified Eagle Medium (DMEM) supplemented with 10% foetal calf serum (FCS), 1% l-glutamine, 1% NSC16168 non-essential amino acids and 0.5% Penicillin/streptomycin, and incubated in a humidified incubator in an atmosphere of 5% CO2 at 37?C. Cell culture conditions for MDA-MB-361, MDA-MB-231 and T47D NSC16168 are defined in [13], CaCo2, HCT116 and MCF7 in [14], Hs578T in [15]. For RNA extraction, cell monolayers were washed with PBS, detached with 0.05% trypsin/EDTA and pelleted for 5?min. Cell pellets were stored in TRI reagent (Sigma-Aldrich) at ?80?C. Table?1 Cell lines used in the experimental study amplification are shown in Supplementary Table?1. All fragments detected by RT-PCR were sequenced (Source BioScience, Nottingham, UK) to confirm specificity and map their position. Quantitative polymerase chain reaction (qRT-PCR) For gene quantification by real-time PCR, Taqman qPCR assays were performed in 20?L reaction volumes containing 10?L Taqman Gene Expression Master Mix (Applied Biosystems), 1?L Taqman gene expression assay and 5?L distilled water. Taqman assays used were Ref. Hs01057642_s1 for and three reference genes (Ref: Hs03044281_g), (Ref: Hs02758991_g1) and (Ref: Hs02800695_m1). qPCR NSC16168 was performed on an Applied Biosystem Fast 7500, with 50 cycles including a hold stage at 94?C for 5?min followed by denaturation step at 94?C for 30?s and then annealing at specific primer temperatures for 45?s, followed by extension at 72?C for 1?min. Statistical analysis For relative gene quantification of mRNA at different time points of neural differentiation, qPCR Ct values were normalised to the geometric mean of those of three reference genes (and value obtained 0.0001, 95% confidence interval, error bars represent RQ, Statistical software Graphpad prism 6 was used: ***sequence across different species. For RNA sequencing analysis, publicly available datasets were downloaded from the European nucleotide archive (ENA, Paired-end RNA-seq [non-stranded, Poly(A)-enriched] was obtained in biological triplicates for a neural differentiation Rabbit Polyclonal to CADM4 from H1 human neural progenitors on time 0, 1, 2, 4, 5, 11 and 18 (Array Express: E-GEOD-56785) [34]. After trimming the info using Trimmomatic [35] (initial 10?bp, quality trimming), the reads were mapped towards NSC16168 the individual guide (Ensembl GRCh38) using HISAT2 [36]. For every test, the transcriptome was constructed using StringTie [37] imposing.