title: Illumina HiSeq 2500 sequencing; Galectin-8 downmodulates TLR4 activation and impairs bacterial clearance in a mouse model of Pseudomonas keratitis
design: Galectin-8 downmodulates TLR4 activation and impairs bacterial clearance in a mouse model of Pseudomonas keratitis
library_name: WT 2_s
library_construction_protocol: FACS sorted neutrophils from infected corneas of wild type (WT) or Gal8 knockout (KO). PA infected corneas were harvested from wild type (WT) or Gal8 knockout (KO, Gal-8-/-) mice on day 1 p.i., pooled group wise (20-30 corneas/group), and digested in 200 µl of DMEM containing collagenase and DNase (100 µg/mL each, Roche Diagnostics, Indianapolis, IN) for 45 min at 37°C in a water bath. After incubation, the corneas were disrupted by grinding with a syringe plunger on a cell strainer, and single-cell suspensions were made in complete DMEM medium. For PMN sorting, cells were blocked with an unconjugated anti-CD32/CD16 mAb (clone 2.4G2, 30 min) in staining buffer (2% FBS, 5 mM EDTA in PBS) and then incubated for 30 min at 4°C with a cocktail of: CD45 (Clone 30-F11), CD11b-PerCP (clone M170), and Ly6G (clone 1A8). All antibodies were purchased from BD Bioscience (San Jose, CA). Then cells were washed three times using PBS and incubated with APC-fixable viability dye (1/1000, 30 min, 4°C), washed again and resuspended for FACS sorting on FACS Aria cell sorter (Becton Dickinson). The yield of neutrophils was 1-1.5 x 10^5 cells/20 infected corneas. RNA extracted using Qiagen RAN extraction assay. ® RNA‑Seq System (NuGEN Technologies) was used to construct cDNA libraries from total RNA.