Dataset Information

Expression of RNA-seq in lesions of a human with tree-man syndrome

ABSTRACT: Two tree-man syndrome lesions immersed in RNA later were snap-frozen in liquid nitrogen and crushed with a tissue pulverizer kit (Cell crusher) according to the manufacturer’s instructions. Total RNA was extracted with the RNeasy Fibrous Tissue Mini Kit (QIAGEN), according to the manufacturer’s instructions. The procedure included a DNAse digestion step or an additional column to remove contaminating DNA. The concentration and purity of the total RNA extracted were measured by spectrometry with an Xpose (Trinean). RNA integrity was evaluated by capillary electrophoresis with a Tape Station (Agilent). The Ovation Universal RNA-Seq System from NUGEN was used to prepare the RNA-seq libraries from100 ng of total RNA, as recommended by the manufacturer. This type of RNAseq kit is less sensitive to total RNA degradation (the RNA integrity number (RIN) of the two samples was quite low: 3.9 for one sample and 5 for the other). This kit constructs strand-specific RNA-seq libraries from 10 to 100 ng of total RNA and uses Insert-Dependent Adaptor Cleavage(InDA-C) technology to remove the ribosomal RNA transcripts. The RNA is subjected to reverse transcription and the second strand is synthesized and a fragmentation step is then performed before Illumina-compatible indexed adaptator ligation. This ligation is followed by a strand-selection enzymatic reaction to provide information about the sense of the transcripts. Insert-dependent adaptor cleavage (InDA-C)-specific primers were then used for the targeted depletion of human ribosomal RNA sequencing transcripts before PCR enrichment. We ensure that no excess amplification occurred during the final PCR step, by evaluating the number of PCR cycles to be applied to each sample in a preliminary Q-PCR test with EvaGreen. An equimolar pool of the final indexed RNA-Seq libraries was sequenced on an Illumina NovaSeq6000 (paired-end reads, 100 bases + 100 bases) and ~50 million paired-end reads per library were produced.

ORGANISM(S): Human papillomavirus type 2 Homo sapiens

PROVIDER: GSE139259 | GEO | 2021/08/11

REPOSITORIES: GEO

ACCESS DATA

Similar Datasets

Project description:The discovery of small open reading frames (smORFs) encoding for polypeptides (SEPs; <100aa) highlights that the coding capacity of the genomes has been underestimated. Most ORF-finding algorithms have historically set a minimum threshold length of 100 aa. As consequence, some transcripts encoding for SEPs, were erroneously discarded or classified as non-coding RNAs (ncRNAs). With this experiments we try to experimentally assess the existence and complexity of these small proteins. The experimental design includes: After growing each Mycoplasma for 6h at 37°C, cells were washed twice with PBS and lysed with 700 µl of Qiazol buffer. Then, samples were lysed with 700 µl of Qiazol buffer. RNA extractions were performed by using the miRNeasy mini Kit (Qiagen) following the instructions of the manufacturer. Libraries for RNA-seq were prepared following directional RNA-seq library preparation and sequencing. Briefly, 1 µg of total RNA was fragmented to ~100-150 nt using NEB Next Magnesium RNA Fragmentation Module (ref. E6150S, NEB). Treatments with Antarctic phosphatase (ref. M0289S, NEB) and PNK (ref. M0201S, NEB) were performed in order to make the 5’ and 3’ ends of the RNA available for adapter ligation. Samples were further processed using the TruSeq small RNA Sample Prep Kit (ref. RS-200-0012, Illumina) according to the manufacturer's protocol. In summary, 3’ adapters and subsequently 5’ adapters were ligated to the RNA. cDNA was synthesized using reverse transcriptase (SuperScript II, ref. 18064-014, Invitrogen) and a specific primer (RNA RT Primer) complementary to the 3’ RNA adapter. cDNA was further amplified by PCR using indexed adapters supplied in the kit. Finally, size selection of the libraries was performed using 6% Novex® TBE Gels (ref. EC6265BOX, Life Technologies). Fragments with insert sizes of 100 to 130 bp were cut from the gel, and cDNA was precipitated and eluted in 10 µl of elution buffer. Double-stranded templates were cluster amplified and sequenced on an Illumina HiSeq 2000.

Project description:Briefly, human embryonic stem cells (hESCs) were collected after BrdU pulse and resuspended in nuclei staining buffer (100 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM CaCl2, 0.5 mM MgCl2, 0.1% NP-40, and 2% bovine serum albumin [Sigma-Aldrich] supplemented with 10 μg/mL Hoechst 33258 [Enzo] and 10 μg/mL propidium iodide [Sigma-Aldrich]. Single nuclei were sorted into 5 μL ProFreeze-CDM freeze medium [Lonza] + 7.5% dimethyl sulfoxide in 96-well skirted PCR plates [4titude], based on low propidium iodide (G1 phase) and low Hoechst (BrdU-induced quenching) fluorescence using an Influx cell sorter [BD Biosciences]. Strand-seq libraries were prepared using an Agilent Bravo liquid handling platform as described before (Sanders et al., 2017). For each experiment, 96 libraries were pooled and 250-450 bp-sized fragments were isolated and purified. DNA quality and concentrations were assessed using the High Sensitivity dsDNA kit [Agilent] on the Agilent 2100 Bio-Analyzer and on a Qubit 2.0 Fluorometer [Life Technologies] resp. Single-end sequencing reads from Strand-seq libraries were generated using the HiSeq 2500 or the NextSeq 500 sequencing platform [Illumina; up to 77 bp]. Indexed reads were aligned to the human reference genome (GRCh38) using Bowtie2 68. Only non-duplicate reads with a mapping quality greater than or equal to 10 were further analyzed with the 69 and AneuFinder R-based package; (https://github.com/ataudt/aneufinder). Strand-seq libraries were prefiltered to avoid errors arising from low-quality data. For this, we excluded libraries with less than 25 reads/Mb, >10% background reads, no template strand inheritance, uneven coverage (high number of copy number segments) or libraries from cells in second cell division. Libraries passing these quality criteria served as input for further analysis. AneuFinder was used to locate and classify template strand switch and copy number change breakpoints. In short, following GC corrections and blacklisting of artefact-prone regions, libraries were analyzed using the edivisive copy number calling algorithm with variable width bins (binsize: 100 kb; step size: 40kb) and breakpoint refinement (R = 10, confint = 0.99; other settings as default). Copy numbers for both the Watson (negative) and Crick (positive) strand were called and breakpoints were defined as changes in copy number state. As BAIT and AneuFinder also detect stable chromosomal rearrangements (e.g., inversions), template switching events that occurred at the exact same locations were excluded from the analysis. Computationally localized SCE or copy number alteration events were further manually verified by visual inspection of chromosome ideograms obtained from AneuFinder and BAIT. Aneuploidy, structural and heterogeneity scores were calculated as previously described.

Dataset Information

Expression of RNA-seq in lesions of a human with tree-man syndrome

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure