Nextrera xt kit

Microglia were gated as CD11c^–Ly6G^–CD11b⁺CD45^low cells and enriched from the mouse brain by FACS as described above, and subjected to bulk RNA-seq. RNA extraction, library preparation, and sequencing were performed at the University of Pittsburgh HSCRF Genomics Research Core as we previously described [18 (link)]. Briefly, total RNA was extracted from FACS-sorted cells using a Qiagen RNeasy Plus Micro Kit, and RNA integrity was assessed using the High Sensitivity RNA ScreenTape system on an Agilent 2200 TapeStation. The SMART-Seq HT Kit was used to generate cDNA from 10 ng of total RNA, and the cDNA product was checked by an Agilent Fragment Analyzer system for quality control. The sequencing library was constructed by following the Illumina Nextera XT Sample Preparation Guide. One nanogram of input cDNA was tagmented (tagged and fragmented) and amplified using the Illumina Nextrera XT kit. Sequence libraries of each sample were finally equimolarly pooled and sequenced on an Illumina Nextseq 500 system, using a paired-end 75-bp strategy.

MEER^vvS and MEER^vvR were cultured in vitro as described above, and cDNA was generated using SMART-seq HT kit (Cat # 634456; Clontech) using 1,000 cells. cDNA product was checked by Tape Station D5000 from Agilent Technologies 2200 to make sure cDNA was successfully generated. Library construction was done using Nextrera XT kit # 15031942 from Illumina. 1 ng cDNA was used in a total volume 5 µl. Sequencing was done using NextSeq 500 System. High Output 75 Cycles kit with run Parameter Paired Read 150 cycles (2 × 75).
Sequencing reads were trimmed for adapters and then aligned to Mus musculus reference genome (Mus_musculus_ensembl_v80_Sequence) using CLC Genomics Workbench. Using CLC Genomics Workbench, differential gene expression was found with statistical cut-offs of P value <0.05, max group means >1, and |foldchange| >1.5. Heatmaps were generated using R package pheatmap with log₂ transformed transcript counts per million (trimmed mean of M-values adjusted).
Gene set enrichment analysis (Mootha et al., 2003 (link); Subramanian et al., 2005 (link)) was performed on differentially expressed genes using the Hallmark gene set.

RNA extraction, library preparation, and sequencing were performed at the Health Sciences Sequencing Core at UPMC Children’s Hospital of Pittsburgh, as previously described (43 (link)). CD8⁺CD122⁺CD49d^lo TRLs (500–1000 cells/sample) were isolated by FACS into a plate with lysis buffer. cDNA was generated from cell lysates using the SMART-Seq Ultralow Input RNA Kit (Takara Bio), according to the manufacturer’s instructions. The cDNA product was checked by an Agilent Fragment Analyzer system for quality control. The sequencing library was constructed by following the Illumina Nextera XT Sample Preparation Guide. One nanogram of input cDNA was tagged and fragmented (“tagmented”) and amplified using the Illumina Nextrera XT kit (15031942). Sequence libraries of each sample were finally equimolarly pooled and sequenced on an Illumina Nextseq 500 system, using a paired-end 75-bp strategy. All RNA-seq data are deposited in the NCBI GEO database.