Mol. Cells 2018; 41(2): 134-139
Published online December 12, 2017
https://doi.org/10.14348/molcells.2018.2246
© The Korean Society for Molecular and Cellular Biology
Correspondence to : *Correspondence: daekee@ewha.ac.kr (DL); jkim1964@ewha.ac.kr (JK)
Here, we report isolation of multiple long non-coding RNAs (lncRNAs) expressed tissue-specifically during murine embryogenesis. One of these, subsequently came to be known as
Keywords erythropoiesis, hematopoiesis, long non-coding RNA,
It has been well-established that most of the mammalian genome is transcribed but only 1~2% code for proteins (Djebali et al., 2012; Pertea, 2012). This implies that most of the RNA species are non-coding and that functional assignment has yet to be made for a vast number of genes. The so-called long non-coding RNAs (lncRNAs) featuring mRNA-like structure but without any apparent open reading frame have been shown to play critical functions in various cellular processes including proliferation, differentiation, stress response, senescence, and apoptosis through diverse molecular mechanisms (Perry and Ulitsky, 2016; Schmitz et al., 2016).
LncRNAs also likely regulate erythropoiesis, the process through which red blood cells are produced (Paralkar and Weiss, 2013). Specifically, several recent studies reported isolation and functional analyses of lncRNAs expressed in maturing erythroblasts and involved in erythropoiesis (Alvarez-Dominguez et al., 2014; Hu et al., 2011; Paralkar et al., 2014). These studies used high-throughput sequencing and RNA interference-based functional tests. Specifically, Alvarez-Dominguez and coworkers reported identification of lncRNAs expressed in early erythroid cells from fetal liver of mice using RNA-seq and functionally validated 12 candidates for their role in terminal maturation using specific siR-NAs (Alvarez-Dominguez et al., 2014). Similarly, Paralkar and coworkers performed RNA-seq to define specific lncRNAs in three distinct cell-types, erythroblasts, cultured megakaryocytes derived from mouse fetal liver and megakaryocyte-erythroid precursors from adult bone marrow (Paralkar et al., 2014). Subsequent RNAi-based screen led to multiple candidates with potential role in terminal erythroid differentiation. One of the lncRNAs that emerged from both studies is
Here, we describe independent isolation of
Total RNA was extracted from whole body and telencephalon of mouse fetuses at embryonic day 12.5 (E12.5) using the RNeasy Mini kit (Qiagen). RNA sequencing data sets for whole body and telencephalon were generated using TruSeq RNA Library Preparation kit v2 and Illumina HiSeq 2000 for 101 bp-long pair-end reads. Raw reads were successfully trimmed using Trimmomatic-0.3. Alignment, removing duplication and indexing were sequentially performed using Tophat v2.0.21 and Picard v1.127 based on the reference sequence of mouse ENSEMBL version78 in the GRCm38 assembly. LncRNAs were identified from ENSEMBL biotype status definition. Differentially expressed genes (DEG) between whole body and telencephalon were determined using Cuffdiff v2.2.1, and LncRNAs with differential expression test
RNA
Total RNA was isolated from indicated tissues of wild type and
Gene trap vector was designed for homologous recombination in the 2nd intron in
Cells were stained with APC-conjugated Rat anti-mouse TER119 (BD Pharmingen, 557909) and PE-conjugated Rat anti-mouse CD71 (BD Pharmingen, 553267) and analyzed by flow cytometry using BD LSRFortessa (BD Bioscience) and BD FACSDiva Software (BD Bioscience). Cell sorting was carried out using BD FACSAria after labeling with anti-mouse TER119 and anti-mouse CD71. The nucleic acid content was examined using BD LSRFortessa after labeling cells with thiazole orange (TO; Sigma Aldrich) or with Retic Count (BD Bioscience). Forward scatter (FSC) analysis was carried out to determine the cell size likewise using BD LSRFortessa.
The plan for this study was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Ewha Womans University.
We extracted RNA from whole mouse embryo and from brain at E12.5 and generated RNA-seq data. Based on subsequent DEG analysis, we selected highly differentially expressed lncRNAs from each of the two sources relative to the other leading to 19 tissue-specifically expressed candidates that may function during embryogenesis (
We chose
Next, we sought to determine the stage of erythropoiesis during which
The homozygous mutant mice were born at the Mendelian ratio, grossly normal, and fertile. Cells isolated from fetal livers were examined by flow cytometry to examine erythropoiesis. Compositions of sub-populations representing various stages erythropoiesis based on expression of two surface markers, CD71 and TER119, were virtually identical among embryos of different genotypes. The results indicated that erythropoiesis was largely normal in
We report detailed embryonic expression patterns of multiple lncRNAs in this study. Our RNA
Mol. Cells 2018; 41(2): 134-139
Published online February 28, 2018 https://doi.org/10.14348/molcells.2018.2246
Copyright © The Korean Society for Molecular and Cellular Biology.
Yerim Lee1, Charny Park1,2, Sanghyuk Lee1,2, Daekee Lee1,2,*, and Jaesang Kim1,2,*
1Department of Life Science, Ewha Womans University, Seoul 03760, Korea, 2Ewha Research Center for Systems Biology, Seoul 03760, Korea
Correspondence to:*Correspondence: daekee@ewha.ac.kr (DL); jkim1964@ewha.ac.kr (JK)
Here, we report isolation of multiple long non-coding RNAs (lncRNAs) expressed tissue-specifically during murine embryogenesis. One of these, subsequently came to be known as
Keywords: erythropoiesis, hematopoiesis, long non-coding RNA,
It has been well-established that most of the mammalian genome is transcribed but only 1~2% code for proteins (Djebali et al., 2012; Pertea, 2012). This implies that most of the RNA species are non-coding and that functional assignment has yet to be made for a vast number of genes. The so-called long non-coding RNAs (lncRNAs) featuring mRNA-like structure but without any apparent open reading frame have been shown to play critical functions in various cellular processes including proliferation, differentiation, stress response, senescence, and apoptosis through diverse molecular mechanisms (Perry and Ulitsky, 2016; Schmitz et al., 2016).
LncRNAs also likely regulate erythropoiesis, the process through which red blood cells are produced (Paralkar and Weiss, 2013). Specifically, several recent studies reported isolation and functional analyses of lncRNAs expressed in maturing erythroblasts and involved in erythropoiesis (Alvarez-Dominguez et al., 2014; Hu et al., 2011; Paralkar et al., 2014). These studies used high-throughput sequencing and RNA interference-based functional tests. Specifically, Alvarez-Dominguez and coworkers reported identification of lncRNAs expressed in early erythroid cells from fetal liver of mice using RNA-seq and functionally validated 12 candidates for their role in terminal maturation using specific siR-NAs (Alvarez-Dominguez et al., 2014). Similarly, Paralkar and coworkers performed RNA-seq to define specific lncRNAs in three distinct cell-types, erythroblasts, cultured megakaryocytes derived from mouse fetal liver and megakaryocyte-erythroid precursors from adult bone marrow (Paralkar et al., 2014). Subsequent RNAi-based screen led to multiple candidates with potential role in terminal erythroid differentiation. One of the lncRNAs that emerged from both studies is
Here, we describe independent isolation of
Total RNA was extracted from whole body and telencephalon of mouse fetuses at embryonic day 12.5 (E12.5) using the RNeasy Mini kit (Qiagen). RNA sequencing data sets for whole body and telencephalon were generated using TruSeq RNA Library Preparation kit v2 and Illumina HiSeq 2000 for 101 bp-long pair-end reads. Raw reads were successfully trimmed using Trimmomatic-0.3. Alignment, removing duplication and indexing were sequentially performed using Tophat v2.0.21 and Picard v1.127 based on the reference sequence of mouse ENSEMBL version78 in the GRCm38 assembly. LncRNAs were identified from ENSEMBL biotype status definition. Differentially expressed genes (DEG) between whole body and telencephalon were determined using Cuffdiff v2.2.1, and LncRNAs with differential expression test
RNA
Total RNA was isolated from indicated tissues of wild type and
Gene trap vector was designed for homologous recombination in the 2nd intron in
Cells were stained with APC-conjugated Rat anti-mouse TER119 (BD Pharmingen, 557909) and PE-conjugated Rat anti-mouse CD71 (BD Pharmingen, 553267) and analyzed by flow cytometry using BD LSRFortessa (BD Bioscience) and BD FACSDiva Software (BD Bioscience). Cell sorting was carried out using BD FACSAria after labeling with anti-mouse TER119 and anti-mouse CD71. The nucleic acid content was examined using BD LSRFortessa after labeling cells with thiazole orange (TO; Sigma Aldrich) or with Retic Count (BD Bioscience). Forward scatter (FSC) analysis was carried out to determine the cell size likewise using BD LSRFortessa.
The plan for this study was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Ewha Womans University.
We extracted RNA from whole mouse embryo and from brain at E12.5 and generated RNA-seq data. Based on subsequent DEG analysis, we selected highly differentially expressed lncRNAs from each of the two sources relative to the other leading to 19 tissue-specifically expressed candidates that may function during embryogenesis (
We chose
Next, we sought to determine the stage of erythropoiesis during which
The homozygous mutant mice were born at the Mendelian ratio, grossly normal, and fertile. Cells isolated from fetal livers were examined by flow cytometry to examine erythropoiesis. Compositions of sub-populations representing various stages erythropoiesis based on expression of two surface markers, CD71 and TER119, were virtually identical among embryos of different genotypes. The results indicated that erythropoiesis was largely normal in
We report detailed embryonic expression patterns of multiple lncRNAs in this study. Our RNA
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