Single cell and bulk RNAseq profiles of thymic pro-T cells

The single cell and bulk expression profiles of early pro T-cell progenitors from mouse thymus Examination of expression profiles of thymic ETP-DN2 population with bulk RNAseq and single cell RNAseq. 10X: Droplet-based 3' end massively parallel single-cell RNA sequencing droplets were prepared using Chromium Single Cell 3' Reagent Kits v2 according to manufacturer's protocol (10x Genomics). C1: Microfluidic-based full length mRNA scRNAseq were performed with Single-Cell mRNA Seq IFC 5' 10 µm chip, and Smartseq 2 chemistry. Bulk: Bulk RNAseq from FACS sorted early T cell populations were performed. RNAeasy MicroKit (Qiagen) were used to purify the total RNA according to manufacturer's recommendations. Purified RNA samples have been processed with and without SMART-Seq v4 Ultra Low Input RNA Kit (Takara #634888) for pre-amplification, and Nextera® XT library preparation kits for Illumina sequencing. Samples without pre-amp were constructed using NEBNext Ultra RNA Library Prep Kit for Illumina (NEB #E7530) from ~1 ug of total RNA following manufacturer's instructions. Libraries were sequenced on Illumina HiSeq2500 in single read mode with the read length of 50-51 nt following manufacturer's instructions. Base calls were performed with RTA 1.13.48.0 followed by conversion to FASTQ with bcl2fastq 1.8.4 and produced approximately 30 million reads per sample.小鼠胸腺早期前T细胞祖细胞的单细胞与批量表达谱数据集：本研究通过批量RNA测序（bulk RNAseq）与单细胞RNA测序（scRNAseq），对胸腺ETP-DN2细胞群的表达谱展开分析。 10X：基于液滴的3'端大规模并行单细胞RNA测序，实验依照制造商10x Genomics的操作手册，使用Chromium单细胞3'端试剂试剂盒v2制备液滴反应体系。 C1：基于微流控的全长mRNA单细胞RNA测序，采用Single-Cell mRNA Seq IFC 5' 10 µm芯片与Smartseq 2测序化学体系完成实验。 Bulk：针对经荧光激活细胞分选（Fluorescence-Activated Cell Sorting，FACS）分选的早期T细胞群开展批量RNA测序。总RNA纯化采用RNAeasy MicroKit（Qiagen），操作严格遵循制造商推荐的实验方案。纯化后的RNA样本分为两组：一组使用SMART-Seq v4超低起始量RNA试剂盒（Takara #634888）进行预扩增，随后采用Nextera® XT文库制备试剂盒完成Illumina测序文库构建；另一组不进行预扩增，以约1 μg总RNA为起始材料，依照制造商说明书使用NEBNext Ultra RNA文库制备试剂盒（NEB #E7530）构建测序文库。测序在Illumina HiSeq2500平台以单端测序模式完成，读长为50~51 nt。碱基识别由RTA 1.13.48.0软件完成，随后通过bcl2fastq 1.8.4转换为FASTQ格式，每个样本约产生3000万条读段。