GSM9514167: RNA-seq of mouse microglia, MCAO day 28, wild-type; Mus musculus; RNA-Seq
Sham-operated mice or mice after MCAO were transcardially perfused with saline, and then the forebrain was removed. After homogenization with RPMI-1640, 1 mg ml–1 collagenase (Sigma-Aldrich) and 50 mg ml–1 DNase I (Sigma-Aldrich) were added to the homogenates. Brain homogenates were digested for 25 min and subjected to Percoll (GE Healthcare) gradient centrifugation. From the interlayer between 37% and 70% Percoll, cells were isolated and stained with anti-CD45 FITC (30-F11, eBioscience), anti-CD11b PerCP-Cy5.5 (M1/70, eBioscience), and anti-CD31 PE/Cyanine7 (390, eBioscience). The cells were sorted using a FACSAria III cell sorter (BD/Thermo Fisher Scientific). Total RNA was purified using a RNeasy micro kit (Qiagen) and used for the preparation of RNA-seq libraries by an Ovation SoLo RNA-Seq Library Preparation Kit (Tecan), according to the manufacturer's procedure. Following the assessment of quantity and fragment size distribution of cDNA libraries by Agilent TapeStation 4150, the libraries were sequenced by Illumina HiSeq 2500. Libraries for the Day 14 Igf1 cohort were sequenced on Illumina NovaSeq 6000.