Unknown,Transcriptomics,Genomics,Proteomics

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DNA-seq of a library of mutated minigene reporters.

ABSTRACT: We developed a high-throughput mutagenesis screen to comprehensively identify the cis-regulatory elements that control a target splicing event from the MST1R gene that codes for the RON receptor tyrosine kinase. Skipping of alternative exon 11 results in a constitutively active isoform that promotes epithelial to mesenchymal transition and thereby contributes to the invasive phenotype of tumors. First, we created a library of mutated minigenes via mutagenic PCR. Importantly, the reverse primer introduced a random barcode sequence which labels the associated mutations. Next, the plasmid library was transfected as a pool and depending on the mutations, the transcripts exhibit changes in alternative splicing. The minigene library and the splicing outcome were analyzed by next-generation sequencing and subsequent integration of the datasets resulted in a map of splicing regulatory sites. The DNA-seq experiment was performed to map the mutations and the associated barcodes in order to identify all minigene variants in the library. For sequencing, we generated five overlapping amplicons of the minigene library using four different forward primers: 5’CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATCTNNNNNNNNNNCTATAGGGAGACCCAAGCTT 3’, 5’CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATCTNNNNNNNNNNGTTCCACTGAAGCCTGAG 3’, 5’CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATCTNNNNNNNNNNAGCTGCCAGCACGAGTTC 3’, 5’CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATCTNNNNNNNNNNGAATCTGAGTGCCCGAGG 3’, and 5’CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATCTNNNNNNNNNNctactggctggtcctcatga 3’, and 5’AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNNNNNATAGAATAGGGCCCTCTAGA 3’ as a common reverse primer. After amplification, the PCR products were cleaned using the GeneRead size selection Kit (QIAGEN) according to manufacturer’s instructions. The purified products were first analysed with the TapeStation 2200 capillary gel electrophoresis instrument (Agilent) and then fluorimetrically quantified using a Qubit fluorimeter (Thermo Scientific). Sequencing was carried out on the Illumina MiSeq platform using paired-end reads of 300 nt length and a 10% PhiX spike-in to increase sequence complexity.

INSTRUMENT(S): Illumina MiSeq

ORGANISM(S): Homo sapiens

SUBMITTER: Julian König

PROVIDER: E-MTAB-6219 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:We developed a high-throughput mutagenesis screen to comprehensively identify the cis-regulatory elements that control a target splicing event from the MST1R gene that codes for the RON receptor tyrosine kinase. Skipping of alternative exon 11 results in a constitutively active isoform that promotes epithelial to mesenchymal transition and thereby contributes to the invasive phenotype of tumors. First, we created a library of mutated minigenes via mutagenic PCR. Importantly, the reverse primer introduced a random barcode sequence which labels the associated mutations. Next, the plasmid library was transfected as a pool and depending on the mutations, the transcripts exhibit changes in alternative splicing. The minigene library and the splicing outcome were analyzed by next-generation sequencing and subsequent integration of the datasets resulted in a map of splicing regulatory sites. The RNA-seq experiment was performed to quantify the transcript isoforms and sequence their associated barcodes in order to characterize the splicing outcome of the minigene library in HEK293T cells. For preparation of high-throughput RNA sequencing (RNA-seq) libraries, the total RNA obtained from transfected HEK293T cells was enriched for mRNA by performing polyA selection of 20 µg of total RNA using Dynabeads® Oligo (dT)25 beads (Invitrogen) according to the manufacturer’s protocol. Reverse transcription was carried out using 500 ng of enriched mRNA under the abovementioned conditions. To prevent the formation of chimeric amplicons, the libraries were amplified using emulsion PCR (PMID: 16791213), with Phusion DNA Polymerase (NEB) and either cDNA derived from polyA-selected RNA in the case of RNA-seq or plasmid DNA of the minigene library in the case of high-throughput DNA sequencing (DNA-seq). To amplify fragments for RNA-seq, the following primers containing Illumina sequencing adaptors were used: 5′-CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATCTNNNNNNNNNNGTTCCACTGAAGCCTGAG-3′ (forward primer) and 5′-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNNNNNATAGAATAGGGCCCTCTAGA-3′ (reverse primer). PCR products were purified using Agencourt AMPure XP beads (Beckman Coulter). Purified products were first analysed with the TapeStation 2200 capillary gel electrophoresis instrument (Agilent) and then fluorimetrically quantified using a Qubit fluorimeter (Thermo Scientific). Sequencing was carried out on the Illumina MiSeq platform using paired-end reads of 300 nt length and a 10% PhiX spike-in to increase sequence complexity.

Project description:We developed a high-throughput mutagenesis screen to comprehensively identify the cis-regulatory elements that control a target splicing event from the MST1R gene that codes for the RON receptor tyrosine kinase. Skipping of alternative exon 11 results in a constitutively active isoform that promotes epithelial to mesenchymal transition and thereby contributes to the invasive phenotype of tumors. First, we created a library of mutated minigenes via mutagenic PCR. Importantly, the reverse primer introduced a random barcode sequence, which labels the associated mutations. Next, the plasmid library was transfected as a pool and depending on the mutations, the transcripts exhibit changes in alternative splicing. The minigene library and the splicing outcome were analyzed by next-generation sequencing and subsequent integration of the datasets resulted in a map of splicing regulatory sites. The RNA binding protein hnRNP H was identified as an important regulator of RON exon 11 splicing. Thus, we performed RNA-seq experiments from minigene library transfected MCF7 cells under control and hnRNP H knockdown conditions in order to quantify the transcript isoforms originating from the minigene library. For preparation of high-throughput RNA sequencing (RNA-seq) libraries, MCF7 cells were first treated with single small interfering RNA (siRNA) against HNRNPH (5′-GGAGCUGGCUUUGAGAGGA[dT][dT] -3′, PMID: 21512137, Sigma-Aldrich) or non-targeting control siRNA (5′-UGGUUUACAUGUCGACUAA[dT][dT]-3′, Sigma-Aldrich) at a final concentration of 20 nM. Next, cells were transfected with the minigene library the day after. A day later, RNA was extracted and subsequently enriched for mRNA by performing polyA selection of 20 µg of total RNA using Dynabeads® Oligo (dT)25 beads (Invitrogen) according to the manufacturer’s protocol. Reverse transcription was carried out using 500 ng of enriched mRNA under the abovementioned conditions. To prevent the formation of chimeric amplicons, the libraries were amplified using emulsion PCR (PMID: 16791213), with Phusion DNA Polymerase (NEB) and cDNA derived from polyA-selected RNA. To amplify fragments for RNA-seq, the following primers containing Illumina sequencing adaptors were used: 5′-CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATCTNNNNNNNNNNGTTCCACTGAAGCCTGAG-3′ (forward primer) and 5′-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNNNNNATAGAATAGGGCCCTCTAGA-3′ (reverse primer). PCR products were purified using Agencourt AMPure XP beads (Beckman Coulter). Purified products were first analysed with the TapeStation 2200 capillary gel electrophoresis instrument (Agilent) and then fluorimetrically quantified using a Qubit fluorimeter (Thermo Scientific). Sequencing was carried out on the Illumina MiSeq platform using paired-end reads of 300 nt length and a 10% PhiX spike-in to increase sequence complexity.

Project description:We used our high-throughput mutagenesis screen to comprehensively identify the cis-regulatory elements that control a target splicing event from the MST1R gene that codes for the RON receptor tyrosine kinase. Skipping of alternative exon 11 results in a constitutively active isoform that promotes epithelial to mesenchymal transition and thereby contributes to the invasive phenotype of tumors. First, we created a library of mutated minigenes via mutagenic PCR. Importantly, the reverse primer introduced a random barcode sequence, which labels the associated mutations. Next, the plasmid library was transfected as a pool and depending on the mutations, the transcripts exhibit changes in alternative splicing. The minigene library and the splicing outcome were analyzed by next-generation sequencing and subsequent integration of the datasets resulted in a map of splicing regulatory sites. The RNA binding proteins HNRNPH1, PRPF6, PUF60, SMU1 and SRSF2 were identified as regulators of RON exon 11 splicing. Thus, we performed RNA-seq experiments from minigene library transfected HEK293T cells under control and five knockdown conditions in order to quantify the transcript isoforms originating from the minigene library. For preparation of high-throughput RNA sequencing (RNA-seq) libraries, HEK293T cells were first treated with small interfering RNA (siRNA) against HNRNPH1, PRPF6, PUF60, SMU1 and SRSF2 or a non-targeting control siRNA. Next, cells were transfected with the minigene library the day after. A day later, RNA was extracted and subsequently enriched for mRNA by performing polyA selection of 20 µg of total RNA using Dynabeads® Oligo (dT)25 beads (Invitrogen) according to the manufacturer’s protocol. Reverse transcription was carried out using 500 ng of enriched mRNA under the abovementioned conditions. To prevent the formation of chimeric amplicons, the libraries were amplified using emulsion PCR (PMID: 16791213), with Phusion DNA Polymerase (NEB) and cDNA derived from polyA-selected RNA. To amplify fragments for RNA-seq, the following primers containing Illumina sequencing adaptors were used: 5′-CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATCTNNNNNNNNNNGTTCCACTGAAGCCTGAG-3′ (forward primer) and 5′-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNNNNNATAGAATAGGGCCCTCTAGA-3′ (reverse primer). PCR products were purified using Agencourt AMPure XP beads (Beckman Coulter). Purified products were first analysed with the TapeStation 2200 capillary gel electrophoresis instrument (Agilent) and then fluorimetrically quantified using a Qubit fluorimeter (Thermo Scientific). Sequencing was carried out on the Illumina MiSeq platform using paired-end reads of 300 nt length and a 10% PhiX spike-in to increase sequence complexity.

Project description:Background RNA sequencing (RNA-seq) is a powerful technique for identifying and quantifying transcription and splicing events, both known and novel. However, given its recent development and the proliferation of library construction methods, understanding the bias it introduces is incomplete but critical to realizing its value. Results Here we present a method, in vitro transcription sequencing (IVT-seq), for identifying and assessing the technical biases in RNA-seq library generation and sequencing at scale. We created a pool of > 1000 in vitro transcribed (IVT) RNAs from a full-length human cDNA library and sequenced them with poly-A and total RNA-seq, the most common protocols. Because each cDNA is full length and we show IVT is incredibly processive, each base in each transcript should be equivalently represented. However, with common RNA-seq applications and platforms, we find ~50% of transcripts have > 2-fold and ~10% have > 10-fold differences in within-transcript sequence coverage. Strikingly, we also find > 6% of transcripts have regions of high, unpredictable sequencing coverage, where the same transcript varies dramatically in coverage between samples, confounding accurate determination of their expression. To get at causal factors, we used a combination of experimental and computational approaches to show that rRNA depletion is responsible for the most significant variability in coverage and that several sequence determinants also strongly influence representation. Conclusions In sum, these results show the utility of IVT-seq in promoting better understanding of bias introduced by RNA-seq and suggest caution in its interpretation. Furthermore, we find that rRNA-depletion is responsible for substantial, unappreciated biases in coverage. Perhaps most importantly, these coverage biases introduced during library preparation suggest exon level expression analysis may be inadvisable. 5 rRNA-depleted samples with duplicates, 1 polyA selected, 1 total RNA, and 1 plasmid library all without replicates.

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DNA-seq of a library of mutated minigene reporters.

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