GSM8940301: 154700-m-iso_mid; Mus musculus; RNA-Seq
Microglia isolation was performed using the MACS system (Miltenyi Biotec) as described previously. However, no CD11b enrichment step was performed as the intrinsic mKate2 reporter is only expressed in TREM2 expressing (microglia) cells. Hence, FACS was performed based on the mKate2 fluorescence signal and sorted into low, mid and high expressing subpopulations, as a proxy for the TREM2 expression of individual cells. The sorted fractions were spun down and the pellets stored at -80°C before being processed for further analysis. RNA isolation was performed using the RNeasy Micro kit (Qiagen), following manufacturer's instruction. For the RNA sequencing, both the quantity and integrity of RNA were assessed using the HS RNA assay on a Tapestation 4200 system from Agilent. The Smart‐seq2 protocol, as detailed by Picelli et al. (2014), was employed to create non-strand-specific, full transcript sequencing libraries. In summary, 2 µL of total RNA were transferred to a buffer containing 0.2% TritonX‐100, protein-based RNase inhibitor, dNTPs, and oligo‐dT oligonucleotides for priming the subsequent RT reaction on polyadenylated mRNA sequences. The SMART RT reaction was conducted at 42°C for 90 min using commercial SuperScript II (Invitrogen) and a Template-Switching Oligo. The pre-amplification PCR with 17 cycles was carried out to produce double‐stranded DNA from the cDNA template. After normalization, 100-300 pg of amplified cDNA underwent tagmentation and enrichment using self-loaded Tn5 transposase (Diagenode) to construct the final sequencing libraries. Quantification of libraries was performed using the Qubit HS dsDNA assay, and the distribution of library fragment sizes was determined using the D1000 assay on a Tapestation 4200 system (Agilent). The sequencing was executed in single-end mode (75 cycles) on a NovaSeq 6000 System (Illumina) with a NovaSeq 6000 S2 Reagent Kit v1.5 (300 cycles) chemistry. Raw sequencing data were demultiplexed using bcl2fastq2 v2.20 and aligned against M. musculus reference genome (M16) using Kallisto 0,48.