Illumina HiSeq 4000 sequencing; RNA-seq of mouse alveolar macrophages sorted from CD11cCre/+ Tgfbr2fl/fl and control Tgfbr2fl/fl neonatal mice
RNA-seq of mouse alveolar macrophages sorted from CD11cCre/+ Tgfbr2fl/fl and control Tgfbr2fl/fl neonatal mice
Sample 6_s
Mice were sacrificed by CO2 inhalation and intracardially perfused with approximately 20 ml PBS. The lung was carefully removed and cut into small pieces using scissors, followed by a tissue digestion in 0.4 mg/ml collagenase type IV (from Clostridium histolyticum) in HBSS (containing Ca2+ / Mg2+) supplemented with 10 % FCS for 45 min at 37C while agitating. Digested tissues were passed through an 18 G syringe and filtered through a 70m cell strainer to obtain a homogeneous cell suspension. After PBS washing and centrifugation at 4C for 5 min at 1500rpm, the cell pellet was resuspended in red blood cell lysis buffer and incubated on ice for 8 mins. After further PBS washing and centrifugation, the cell pellet was resuspended in antibody mix and stained at 4C for 25 mins. After PBS washing and centrifugation, resuspend the cell pellet in FACS buffer (2mM EDTA, 2%FCS in PBS) and ready for FACS sorting.  Sorted cells were lysed in RLT plus buffer (Qiagen) supplemented with 1 % mercaptoethanol. RNA isolation was performed using the RNeasy Micro Plus Kit (Qiagen) according to the manufacturers instructions. The quantity and quality of the isolated RNA was determined with a Qubit® (1.0) Fluorometer (Life Technologies, California, USA) and a Bioanalyzer 2100 (Agilent,Waldbronn, Germany). The TruSeq Stranded mRNA Sample Prep Kit (Illumina, Inc,California, USA) was used in the succeeding steps. Briefly, total RNA samples (100 ng) were poly-A selected and then reverse-transcribed into double-stranded cDNA with Actinomycin added during first-strand synthesis. The cDNA samples was fragmented, end-repaired and adenylated before ligation of TruSeq adapters. The adapters contain the index for multiplexing. Fragments containing TruSeq adapters on both ends were selectively enriched with PCR. The quality and quantity of the enriched libraries were validated using Qubit® (1.0) Fluorometer and the Bioanalyzer 2100 (Agilent, Waldbronn, Germany). The product is a smear with an average fragment size of approximately 360 bp. The libraries were normalized to 10nM in Tris-Cl 10 mM, pH8.5 with 0.1% Tween 20.