GSM6948192: 4M_5XFAD_rep2; Mus musculus; RNA-Seq
Microglia were sorted directly into ~10uL of TCL buffer (QIAGEN) containing 1% β-mercaptoethanol for RNA extraction. Pre library construction, samples were thawed briefly at RT and ~10uL of each was transferred to a 96-well plate. The construction of the full-length RNA-seq libraries followed a modified SMART-Seq2 protocol81. After first strand cDNA synthesis and amplification, the cDNA was purified with a 0.7x single sided SPRI. The cDNA quantification of samples was done via a Qubit hsDNA assay (Thermo Fisher) and fragment size distribution was assessed with a BioAnalyzer HS dsDNA assay (Agilent). Each sample was normalized to 0.20ng/uL and then tagmented and indexed using the Nextera XT DNA library prep kit (Illumina). The individually barcoded libraries were then pooled and a final quality control was done on the BioAnalyzer (Agilent). The pool was normalized to 4nM, denatured using 0.2 N NaOh and 0.2 M Tris-HCL and diluted in HCL to prepare for sequencing. 1300uL of the final diluted pooled library was loaded for sequencing on a NextSeq 500/550. Paired end sequencing was performed using an Illumina 75 cycle Nextseq 500 high output V2 kit in accordance with Illumina protocol (Reads 38 cycles x2, Index 8 cycles x2). This process was repeated for repeat batches.