title: GSM6132041: LUNG_MAC_NP2; Mus musculus; RNA-Seq
library_construction_protocol: Lung and Skin macrophages and fibroblasts were FACS sorted using a cocktail of fluorescent antibodies (Lung interstitial macrophages: DAPI-CD45+CD68+F4/80+CD11c-CD11bhi; Skin macrophages: DAPI-CD45+CD11b+CD68+F4/80+; Lung fibroblasts: DAPI-CD45-CD31-EPCAM-PDGFRA+;  Skin fibroblasts: DAPI-CD45-CD31-EPCAM-CD90.1+PDGFRA+) with typical purities > 95%. RNA was extracted ussing the RNAeasy plus micro kit from QUIAGEN cDNA amplification from neutrophil RNA and generation of index-tagged sequencing libraries were carried out using the Ovation Single Cell RNA-Seq System (NuGEN Technologies, San Carlos CA). Libraries were quantified using a Quant-iTTM dsDNA HS assay with the Q-bit fluorometer (Life Technologies, Carlsbad, California). Average library size and size distribution were determined using a High Sensitivity DNA assay in an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara CA). Libraries were normalized to 10nM using Tris-Cl 10mM, pH8.5 with 0.1% Tween 20. Libraries were applied to an Illumina flow cell for cluster generation (True Seq SR Cluster Kit V2 cBot) and 61 nt long, single-end reads were generated on a Genome Analyzer IIx, using the TruSeq SBS Kit v5 (Illumina) and following the standard RNA sequencing protocol