title: Illumina HiSeq 2500 paired end sequencing; RNA-seq of primary human M2-polarized macrophages cultured in either high-concentration or low-concentration type I collagen.
design: RNA-seq of primary human M2-polarized macrophages cultured in either high-concentration or low-concentration type I collagen.
library_name: Sample 4_p
library_construction_protocol: Five million M2 macrophages were cultured in a 500 μl three-dimensional gel made of bovine skin collagen (Nutragen, Advanced BioMatrix, 1.7 or 4.8 mg/ml) in RPMI-1640 with 1% fetal bovine serum and 100 ng/ml M-CSF (Peprotech) for 48 hours in a 24-well plate. Gels from two wells were transferred into one 2 ml tube and homogenized with Tissue Tearor (BioSpec Products) in 600 μl of RNA extraction solution (60% v/v water-saturated phenol, 3.25 M guanidine thiocyanate, 400 mM sodium acetate buffer pH 4.0, 0.4% w/v N-lauroylsarcosine, 160 mM 2-mercaptoethanol) plus 100 μl of 6 M sodium chloride. Primary human macrophages derived from CD14+ subpopulation of peripheral blood mononuclear cells from healthy donors were differentiated and M2-polarized with M-CSF (100 ng/ml) in RPMI-1640 medium supplemented with 10% fetal bovine serum for 5 - 6 days before the experiments. 200 μl of chlorophorm was added to approx. 1 ml of the lysate and the mixture was vortexed vigorously for 10 seconds. After 30-minute centrifugation at 18,000 g at 4°C, the polar upper phase was transferred to a new tube, volume adjusted to 800 μl with RNase-free water, RNA was precipitated with 600 μl of isopropanol at -20°C overnight and recovered by centrifugation at 18,000 g for 30 minutes. The RNA pellet was washed three times with 600 μl of 75% ethanol and air-dried. Next, the RNA was treated with DNAse I to remove possible genomic DNA contamination. To this end, the pellet was directly dissolved in 100 μl of solution containing 4 units of DNAse I (Thermo Fisher Scientific) in manufacturer-provided reaction buffer and incubated at 37°C for 30 minutes. After that, the RNA was re-purified with RNeasy Protect Mini Kit (Qiagen) according to the manufacturer's instructions and eluted in 50 μl of RNase-free water.  Stranded, Illumina HiSeq-compatible library was constructed with ScriptSeq Complete (Human/Mouse/Rat) library preparation kit (Epicentre) according to the manufacturer's instructions.
attributes.tag: Experimental Factor: growth condition
attributes.value: high concentration bovine skin collagen; 4.8 milligram per milliliter