title: GSM5099750: month6_ccl4_rep2; Mus musculus; RNA-Seq
library_construction_protocol: RNA was isolated using the TRIzol method. Briefly, 1 ml TRIzol was added to 5 x 106 cells followed by vortexing, a 5-min incubation at room temperature, and the addition of 200 μl chloroform. After mixing, further incubation at room temperature for 2–3 min and centrifugation (12,000 g) at 4 °C for 5 min, the clear supernatant was mixed with 500 μl isopropanol and incubated at room temperature for 10 min. After centrifugation (12,000 g) at 4 °C for 10 min, the supernatant was discarded and the pellet washed with 1 ml cold 75% ethanol followed by vortexing and centrifugation (7,500 g, 4 °C, 5 min). The pellet was dried and dissolved in RNase-free water. For the preparation of the libraries, rRNA was removed with the Ribo-Zero™ Human/Mouse/Rat rRNA Removal Kit from Biozym Scientific GmbH as described in Cadenas et al. (under review). Briefly, the RNA library was prepared using the TruSeq Stranded totalRNA Sample Prep Kit from Illumina (San Diego, CA) according to the manufacturer's instructions. All samples were sequenced using an Illumina HiSeq 2500 sequencer v4 (Illumina, San Diego,CA) with 10 transcriptome samples per lane and an average of 25 million paired-end reads (2×125 bp) at IKMB NGS core facilities. Reads were mapped to the corresponding reference genome using Biojupies.