GSM5605559: RNA-seq Day 0 WT replicate 3A; Mus musculus; RNA-Seq
Mouse spleens, livers, lungs, lymph nodes, and blood were harvested and prepared into single cell suspensions as described previously11. Splenic single cell suspensions were lysed in red blood cell lysis buffer and resuspended in EasySep™ buffer (Stemcell). To avoid depleting Ly6C+ NK cells we developed a custom antibody cocktail as follows:  splenocytes were labeled with 10 μg per spleen of biotin conjugated antibodies against CD3 (17A2), CD19 (6D5), CD8 (53-6.7), CD88 (20/70), Ly6G (1A8), SiglecF (S17007L), TCRβ (H57-597), CD20 (SA275A11), CD172a (P84) and magnetically depleted from total splenocyte suspensions with the use of anti-biotin coupled magnetic beads (Biolegend). RNA was isolated from the cells using RNeasy Mini kit (Qiagen) and used to generate RNA-seq libraries followed by sequencing using Illumina HiSeq 4000 platform (single end, 50bp). The reads were mapped with HISAT2 (version 2.2.1)  to the mouse genome (mm10).  The counts for each gene were obtained by HtSeq (Simon Anders, Paul Theodor Pyl, Wolfgang Huber HTSeq — A Python framework to work with high-throughput sequencing data Bioinformatics (2014), in print, online at doi:10.1093/bioinformatics/btu638). Differential expression analyses were carried out using DESeq257  (version 1.24.0) with default parameters. Genes with adjusted p value <0.05 were considered significantly differentially expressed. Sequencing depth normalized counts were used to plot the expression values for individual genes. For ATAC-Seq, 50,000 cells per sample were lysed to collect nuclei and treated with Tn5 transposase (Illumina) for 30 minutes at 37°C with gentle agitation. The DNA was isolated with DNA Clean & Concentrator Kit (Zymo) and PCR amplified and barcoded with NEBNext High-Fidelity PCR Mix (New England Biolabs) and unique dual indexes (Illumina). The ATAC-Seq library amplification was confirmed by real-time PCR, and additional barcoding PCR cycles were added as necessary while avoiding overamplification. Amplified ATAC-Seq libraries were purified with DNA Clean & Concentrator Kit (Zymo). The purified libraries were quantified with Kapa Library Quant Kit (KAPA Biosystems) and quality assessed on 4200 TapeStation System (Agilent). The libraries were pooled based on molar concentrations and sequenced on an Illumina HiSeq 4000 platform (paired end, 100bp).