Data describing the effects of Induced knockout of Heterogeneous nuclear ribonucleoprotein K in mouse skeletal muscle satellite cells

HnRNPK, a prominent member of the heterogeneous nuclear ribonucleoprotein (hnRNP) family, is widely expressed in mammalian tissues and plays a crucial role in animal development. Despite its well-established functions, limited information is available regarding its role in skeletal muscle development and regeneration. To elucidate the functional role of hnRNPK in skeletal muscle, we utilized Pax7CreER; HnrnpkLoxP/LoxP (Hnrnpk pKO) mice as a model, isolated primary mouse skeletal muscle satellite cells (MuSCs), and induced hnRNPK knockout using 4-OTH. Transcriptome sequencing was performed on four distinct groups: Hnrnpk pKO MuSCs undergoing proliferation for 24 h (ethanol 24 h) and 48 h (ethanol 48 h) after treatment with ethanol as the control, as well as Hnrnpk pKO MuSCs undergoing proliferation for 24 h (4-OHT 24 h) and 48 h (4-OHT 48 h) after treatment with 4-OHT as the hnRNPK-induced knockout group. The RNA sequencing data was generated using the Illumina HiSeq 2000/2500 sequencing platform. The raw data files have been archived in the Sequence Read Archive at the China National Center for Bioinformation (CNCB) under the accession number CRA015864. This data article is related to the research paper “Deletion of heterogeneous nuclear ribonucleoprotein K in satellite cells leads to inhibited skeletal muscle regeneration in mice, Genes & Diseases 11: 101,062, DOI: 10.1016/j.gendis.2023.06.031”.

a b s t r a c t HnRNPK, a prominent member of the heterogeneous nuclear ribonucleoprotein (hnRNP) family, is widely expressed in mammalian tissues and plays a crucial role in animal development.Despite its well-established functions, limited information is available regarding its role in skeletal muscle development and regeneration.To elucidate the functional role of hnRNPK in skeletal muscle, we utilized Pax7 CreER ; Hnrnpk LoxP/LoxP ( Hnrnpk pKO) mice as a model, isolated primary mouse skeletal muscle satellite cells (MuSCs), and induced hnRNPK knockout using 4-OTH.Transcriptome sequencing was performed on four distinct groups: Hnrnpk pKO MuSCs undergoing proliferation for 24 h (ethanol 24 h) and 48 h (ethanol 48 h) after treatment with ethanol as the control, as well as Hnrnpk pKO MuSCs undergoing proliferation for 24 h (4-OHT 24 h) and 48 h (4-OHT 48 h) after treatment with 4-OHT as the hnRNPK-induced knockout group.The RNA sequencing data was generated using the Illumina HiSeq 20 0 0/250 0 sequencing platform.

Value of the Data
• The dataset contains the transcriptomes of MuSCs proliferation growth after induced knockout of hnRNPK, serving as a reference for studying hnRNPK's roles and downstream genes.
• The dataset provides key information for a comparative analysis of differentially expressed genes between the MuSCs and hnRNPK knockout MuSCs.

• Analysis of gene expression changes in proliferation growth of MuSCs and hnRNPK knockout
MuSCs will help investigate the importance of hnRNPK in self-renewal and fate of MuSCs.• In addition, the dataset can be used to examine the functional analysis of genes regulated by hnRNPK in MuSCs proliferation, and also it could provide very useful information to reveal the mechanism of action of hnRNPK in mouse muscle regeneration.

Background
A growing body of research on hnRNPK indicates its significance in animal development, cell proliferation, and differentiation via transcriptional and post-transcriptional regulation [ 1 ].Nonetheless, there is a paucity of studies on its involvement in skeletal muscle development, and the downstream target genes remain unidentified.To address this gap, we generated Hnrnpk mKO by utilizing Hnrnpk LoxP/LoxP in conjunction with Myf5 -Cre mice.The Hnrnpk mKO mice exhibited mortality prior to 18.5 dpc, with surviving 17.5 dpc mice showing significant absence of muscle tissue (unpublished data), underscoring the essential role of hnRNPK in skeletal muscle development.Subsequently, we generated inducible satellite cell specific knockout mice utilizing Hnrnpk LoxP/LoxP and Pax7 CreER mice.The induced knockout of hnRNPK in mice severely impeded muscle regeneration.Mechanistically, the interaction between hnRNPK and Cdkn1a mRNA was implicated in these processes 2 ].However, considering the complexity functions of hnRNPK, its target gene is not only Cdkn1a , but may also regulate MuSCs growth through other target genes.The induced knockout of hnRNPK in MuSCs was used for RNA sequencing to obtain differentially expressed genes by altered expression of hnRNPK.The resulting transcriptome data can be useful to identify potential downstream genes of hnRNPK that involved in skeletal muscle development and regeneration .

Animal used
All animal experiments in this study were approved by the Institutional Review Board of Xinyang Normal University (Xinyang, China).Mice were hosted on a 12 h light/12 h dark cycle (8:0 0AM to 8:0 0PM) at humidity of 50 ∼70 % and 22 ± 2 • C ambient temperature with free access to food and water.Hnrnpk LoxP/LoxP mice were generated with the aid of Cyagen Biosciences Inc. (Suzhou, Jiangsu, China) in a C57BL/6 genetic background.Genotypes were identified by PCR analysis as previously described [ 3 ].Skeletal muscle satellite cell-specific Hnrnpk knockout mice were generated according to the strategy as follows.Briefly, inducible mouse model Pax7 CreER (The Jackson Laboratory, stock no.012,476) mice were crossed with Hnrnpk LoxP/LoxP mice to obtain Pax7 CreER ; Hnrnpk LoxP/ + mice, which were crossed with adult Hnrnpk LoxP/LoxP mices, and the Pax7 CreER ; Hnrnpk LoxP/LoxP male mice were considered to be the conditional homozygous knockout mice ( Hnrnpk pKO), and the littermates Hnrnpk LoxP/LoxP mice were used as the wild-type (WT) control.Mice were genotyped by PCR using standard protocols.

MuSCs isolation and culture
MuSCs were isolated using type I collagenase and dispase B digestion as previously described [ 4 ].Briefly, the hind limb skeletal muscles from the Hnrnpk pKO mice were collected, minced, and digested in 2.5 ml of collagenase/dispase solution (10 μg/ml collagenase B, 2.4 units/ml dispase B in PBS) for 1 h.The digestions were stopped with RPMI 1640 medium containing 20 % fetal bovine serum and centrifuged at 10 0 0 g for 10 min.Then the cells were seeded on Matrigel-coated dishes and cultured in growth medium containing RPMI 1640 medium, with 20 % fetal bovine serum, 2.5 ng/ml bFGF, 0.5 % CEE, 1 % GlutaMAX, 1 % NEAA, and 1 % penicillinstreptomycin at 37 °C with 5 % CO 2 .The medium was changed every 2 days.MuSCs were used for analysis after purification by 2-3 times of pre-plating.To induce MuSCs differentiation, the culture medium was replaced with DMEM supplemented with 2 % horse serum (Gibco, USA) and 1 % PS at 80 % confluence.To induce the knockout of hnRNPK in Hnrnpk pKO MuSCs were treated with 40 nm 4-hydroxytamoxifen (4-OHT) (Sigma) for 48 h, and treated with ethanol as control.Cells were then used for the analysis after removal of the 4-OHT or ethanol.

RNA-Seq library preparation and analysis
RNA was isolated from hnRNPK induced knockout MuSCs and control MuSCs at proliferation 24 or 48 h after 4-OHT or ethanol treatment using RNAisoPlus reagent (TaKaRa, Japan).The qualified total RNA was further cleaned up by using RNAClean XP Kit (Beckman, Krefeld, Germany) and RNase-Free DNase Set (QIAGEN, Hilden, Germany).After passing through the quality inspection, the RNA-Seq library was constructed using the VAHTS Stranded mRNA-seq Library Prep Kit for Illumina® (Vazyme, NR602-02), and sequenced on the Illumina Hiseq 20 0 0/250 0 NextSeq with paired-end sequencing in Shanghai Bohao Biotechnology Co., Ltd.After prefiltering the raw data by removing the joint sequence and low-quality reads, the preprocessed reads were conducted to align the mouse genome GRCm38.p4(mm10) using Hisat2 estimated by (version: 2.0.4) alignment software for genome mapping [ 5 ].The expression level of each gene was normalized by FPKM (Fragments per Kilobase of exon model per Million mapped reads) using StringTie software (version 1.3.0)[ 6 ].The gene differential expression was determined using EdgeR ver.3.40.2[ 7 ], and the statistical analysis of differential expressed gene (DEG) was calculated by DESeq2 ver.1.12.4 software with a Wald's test [ 8 ].Genes exhibiting a log2Fold Change ≥ 1.0 or log2Fold Change ≤ −1.0 and Q-value ≤ 0.05 were considered to be significantly differentially expressed in the testes derived from 4-OTH group compared to in ethanol control groups, and growth 48 h group compared to 24 h group.
The raw data files have been archived in the Sequence Read Archive at the China National Center for Bioinformation (CNCB) under the accession number CRA015864.This data article is related to the research paper "Deletion of heterogeneous nuclear ribonucleoprotein K in satellite cells leads to inhibited skeletal muscle regeneration in mice, Genes & Diseases 11: 101,062, DOI: 10.1016/j.gendis.2023.06.031".© 2024 The Authors.Published by Elsevier Inc.

Table 1
Summary of RNA sequencing data.