Transcriptomic atlas reveals organ-specific disease tolerance in sickle cell mice: dataset bone marrow HbAA mice injected or not with heme

The objective of this experiment was to explore the transcriptome of the HbSS Townes mouse model of sickle cell disease. Townes model mice carry several human hemoglobin knock-in genes replacing the endogenous mouse genes and may be useful in studying sickle cell disease. All mice were genotyped, age- and sex-matched littermates. All HbAA (control, normal human hemoglobin) vs HbSS (sickle cell disease, mutated human hemoglobin) mice were used for experimentations at 6-8 weeks of age, to limit intra-group heterogeneity. Hemin (Ferriprotoporphyrin IX) was purchased from Frontiers Scientific and injected intravenously (iv.) in a retroorbital sinus at a concentration of 24 µmol/kg. Control mice received PBS instead. Mice were anesthetized with isoflurane 2-3% for injections, blood collection and sacrifice. All mice were sacrificed by cervical dislocation, 4 hours after injection. This dataset contains the results of the HbAA mice with and without heme. The corresponding HbSS mice with and without heme are deposited under number 10.5281/zenodo.10962782 Bone marrow RNA was extracted by Macherey Nagel kit, according to the manufacturer’s instructions. The quality and quantity of mRNA were evaluated using a 2100bioanalyzer with TNA 6000 NanoKits (all Agilent Technologies, Palo Alto, CA, USA). RNA Integrity Numbers superior to 7 were eligible for subsequent reverse transcription into cDNA. RNAseq was performed at the GenomIC plateform Cochin Institute INSERM U1016. After RNA extraction, RNA quality (RNA integrity number) was estimated. 1μg of high-quality total RNA sample (RIN >7) was processed to build up the libraries, using TruSeq Stranded mRNA kit (Illumina) according to manufacturer instructions. Briefly, purified poly-A containing mRNA molecules were fragmented and reverse-transcribed using random primers. Replacement of dTTP by dUTP during second strand synthesis allowed us to achieve strand specificity. Addition of a single A base to the cDNA was followed by ligation of Illumina adapters.Libraries were quantified by qPCR using KAPA Library Quantification Kits for Illumina Libraries (KapaBiosystems, Wilmington, MA). Library profiles were assessed using DNA High Sensitivity LabChip kits on an Agilent Bioanalyzer. Libraries were sequenced on an Illumina Nextseq 500 instrument using 75 base-lengths read V2 chemistry in a paired-end mode. After sequencing, primary analysis based on AOZAN software (ENS, Paris), was applied to demultiplex and control the quality of the raw data (based of FastQC modules / version 0.11.5). The dataset here represents 4 groups of mice, 4 mice per group as follows: HbAA PBS, HbAA heme, HbSS PBS, HbSS heme.本实验旨在探究镰状细胞病HbSS Townes小鼠模型的转录组。Townes模型小鼠携带多个人类血红蛋白敲入基因，替代内源性小鼠基因，可用于镰状细胞病的相关研究。所有小鼠均经过基因分型，选用年龄、性别匹配的同窝仔鼠。为限制组内异质性，实验均使用6-8周龄的HbAA（对照组，正常人类血红蛋白）与HbSS（镰状细胞病组，突变型人类血红蛋白）小鼠。 氯化血红素（铁原卟啉IX，Ferriprotoporphyrin IX）购自Frontiers Scientific，经眶后静脉窦以24 μmol/kg的浓度静脉注射。对照组小鼠则注射磷酸盐缓冲液（PBS）替代。注射、采血及处死过程中，小鼠均用2-3%异氟烷进行麻醉。所有小鼠于注射后4小时经颈椎脱臼法处死。 本数据集包含HbAA小鼠伴与不伴血红素处理的实验结果。对应的HbSS小鼠伴与不伴血红素处理的数据集已存于编号10.5281/zenodo.10962782。 骨髓RNA采用Macherey Nagel试剂盒提取，操作严格遵循制造商说明书。使用搭载TNA 6000 Nano试剂盒的2100生物分析仪（均为美国加利福尼亚州帕洛阿尔托安捷伦科技产品）评估mRNA的质量与浓度。RNA完整数（RNA Integrity Number，RIN）大于7的样本方可进行后续的逆转录合成cDNA。RNA测序（RNAseq）在INSERM U1016科钦研究所的GenomIC平台完成。 RNA提取完成后，先对RNA质量（RNA完整数）进行评估。取1μg高质量总RNA（RIN>7），按照制造商说明书使用TruSeq Stranded mRNA试剂盒（Illumina）构建文库。简要步骤如下：纯化含poly-A尾的mRNA分子并进行片段化，随后使用随机引物进行逆转录。在第二链合成过程中用dUTP替代dTTP，以实现链特异性。在cDNA末端添加单碱基A后，连接Illumina接头。文库定量采用KAPA文库定量试剂盒（KapaBiosystems，美国马萨诸塞州威尔明顿），通过qPCR完成。文库的片段分布特征使用安捷伦生物分析仪的DNA High Sensitivity LabChip试剂盒进行评估。文库在Illumina Nextseq 500测序仪上采用75碱基读长的V2测序化学试剂进行双端测序。 测序完成后，基于AOZAN软件（巴黎高等师范学院，ENS）进行初步分析，通过FastQC模块（版本0.11.5）完成原始数据的解多重与质量控制。 本数据集共包含4组小鼠，每组4只，具体分组为：HbAA PBS组、HbAA 血红素处理组、HbSS PBS组、HbSS 血红素处理组。