GSM7817434: Brain, CD3+ SS2 KO B24 mouse 3 scRNAseq; Mus musculus; RNA-Seq
To isolate immune cells, after perfusion, brains were digested for 45 min in RPMI containing 10 mM HEPES, 0.5 mg/ml Collagenase Type IV (Sigma Aldrich) and 0.05 mg/ml DNase I (Sigma Aldrich) at 37°C. Cells were then isolated using density gradient centrifugation. The isolated brain cells were directly sorted on an BD Aria III cell sorter for sequencing experiments. The left hemisphere was used to sort CD45+ cells to perform scRNAseq with SeqWell v3 (Aicher et al. 2019). The right hemisphere was used for index sorting of CD3+ cells into 384 well plates (Penter et al. 2018) for scRNAseq with SmartSeq2 (Picelli et al. 2014). Only cells isolated with a purity of > 98% were used for further analysis. CNS T cells from the brains of B57BL/6 Zfyve26-/- 𝐶𝑋𝐶𝑅4;𝐶𝑟𝑒𝐸𝑅(𝑇2)−𝐼𝑅𝐸𝑆−𝐺𝐹𝑃;𝑅26𝐶𝐴𝐺−𝐿𝑆𝐿−𝑡𝑑𝑇 or Zfyve26+/- 𝐶𝑋𝐶𝑅4;𝐶𝑟𝑒𝐸𝑅(𝑇2)−𝐼𝑅𝐸𝑆−𝐺𝐹𝑃;𝑅26𝐶𝐴𝐺−𝐿𝑆𝐿−𝑡𝑑𝑇 animals were isolated as described above. Total RNA was isolated using the miRNeasy Mini Kit (QIAGEN) and transcriptome analysis was performed by SmartSeq2 (Picelli et al. 2014). Per sample, 500 pg RNA was denatured for 2 min at 95°C in 0.1% Triton X-100 containing 0.05 mM dNTP, 2 μM anchored oligodT (Smart dT30VN) primer and 1 U/μl RNase Inhibitor (Takara). Reverse transcription was carried out at 42°C for 90 min followed by 15 min inactivation at 70°C using 5 U/μl Superscript II reverse transcriptase (Thermo Fisher Scientific) in 1X Superscript II Buffer supplemented with 5 mM DTT, 1 M Betaine, 14 mM MgCl2, 1 μM Template Switching Oligo and 1 U/μl RNase Inhibitor. cDNA was then pre-amplified using KAPA HiFi HotStart ReadyMix (Kapa Biosystems) containing 1 nM ISPCR Primer. Amplification was carried out for 16 cycles consisting of 20 s denaturation at 98°C, 20 s annealing at 67°C and 6 min elongation at 72°C each after an initial denaturing at 98°C for 3 min. The pre-amplification reaction was then purified by using a 0.8:1 ratio of AMPure XP beads (Beckman Coulter). DNA was allowed to bind to beads for 3 min at RT followed by one wash in 80% Ethanol for 30 s. cDNA was then eluted from the beads in 15 μl of water before checking for cDNA quality on a Tapestation 2200 using a HighSensitivity D5000 assay (Agilent Genomics) according to manufacturer's instructions. Tagmentation, PCR amplification and indexing of samples was performed using the Nextera XT DNA Library Preparation Kit (Illumina) according to manufacturer's recommendations. After library preparation, DNA was purified with a 1:1 ratio of AMPure XP beads and sample. Quantification and quality check of libraries was done on a Tapestation 2200 using a HighSensitivity D5000 Assay. Library concentrations were adjusted to 2nM and pooled prior to sequencing SR 75 bp with two 8 bp index reads on an Illumina NextSeq 500 instrument using High Output v2 chemistry. RNA-seq data was demultiplexed using bcl2fastq2 v2.20 and pseudo aligned to Mus_musculus.GRCm38.cdna.all.fa.gz transcriptome using kallisto v0.44.0 (Bray et al. 2016). The sequences of all oligos used for SmartSeq2 can be found in Table S7. All oligos were biotinylated at the 5′ end to avoid the formation of concatemers.