GSM8969678: CHD1_KO_dLN_3; Mus musculus; RNA-Seq
For the CHD1 WT vs. cKO and GR WT vs. cKO experiments, EAE mice were euthanized and perfused intracardially with ice-cold PBS. The brain and spinal cord were finely minced using a razor blade and digested with 2.5 mg/ml collagenase D (Roche Diagnostics) at 37°C for 20 minutes. The digested tissue was then passed through a 70-μm strainer. Mononuclear cells were isolated by density gradient centrifugation using a Percoll (Amersham Biosciences) gradient of 37% and 70%. The mononuclear cells were collected from the interphase for further analysis. 2,000 CNS-infiltrating 2D2+ cells were sorted by FACS into 5 μl of TCL buffer (Qiagen) supplemented with 1% 2-mercaptoethanol (Sigma-Aldrich). At the same time, draining lymph nodes (dLNs) cells were obtained by mechanically dispersing the organs through a 70-μm strainer. 2,000 2D2+ cells were sorted from dLN by FACS into 5 μl of TCL buffer (Qiagen) supplemented with 1% 2-mercaptoethanol (Sigma-Aldrich).  For the in vitro Dex treatment experiment, naive T cells from WT or IL-23R KO mice were differentiated into Th17Inf cells in vitro and treated with or without dexamethasone (Dex) for two days. 2,000 live Th17 cells were sorted by FACS into 5 μl of TCL buffer (Qiagen) supplemented with 1% 2-mercaptoethanol (Sigma-Aldrich). Libraries were prepared using a modified SMART-Seq2 protocol, including RNA secondary structure denaturation at 72 °C for 3 min, reverse transcription with Maxima Reverse Transcriptase (Thermo Fischer Scientific) and whole-transcriptome amplification using KAPA HiFi HotStart ReadyMix 2× (KAPA Biosystems) for 12 cycles. Whole-transcriptome amplification product quality was confirmed with a High Sensitivity DNA Chip run on a Bioanalyzer 2100 (Agilent, G2939BA) and quantified using a Qubit dsDNA HS Assay kit (Thermo Scientific). Libraries were prepared with a Nextera XT DNA Library Preparation kit (Illumina) and sequenced using a high-output V2 75 cycle kit (Illumina) on a NextSeq 500 sequencer (Illumina) with 2 × 38 paired-end reads.