GSM3612621: P7_pre_0; Mus musculus; RNA-Seq
Single cell cardiac tissue suspensions were prepared by mincing hearts and gentle agitation in collagenase 500 units per ml in HBSS solution for 1 hour at 37degC. Samples were passed through a 70um filter, washed and resuspended in 2% FBS/PBS. Samples were incubated with FcR-block, and then labelled with anti-CD45 (Biolegend 103124, 1:200), anti-CD11b (Biolegend 101206, 1:100), anti-Ly6G (Biolegend 127636, 1:200), anti-F4/80 (Biolegend 123110, 1:100), anti-Ly6C (Biolegend 128026 1:800), anti-CD206 (Biolegend 141721, 1:100). Macrophages were identified as CD45+ CD11b+ Ly6G- F4/80+ cells RNA extraction and DNAse treatment were carried out using the RNAqueous®-Micro Kit (Life Technologies, cat # AM1931). RNA integrity was checked with a RNA pico chip (Agilent Technologies, cat # 5067-1513) using the Agilent 2100 Bioanalyzer. cDNA was synthesized and amplified from 100 pg – 1 ng of input RNA using SMART-seqTMv4 ultra low input kit for RNA (Clontech laboratories, cat #'s 634888, 634889, 634890, 634891, 634892, 634893, and 634894). Sequencing libraries were prepared using the Nextera XT DNA library preparation kit and sequencing performed on a NextSeq platform using a NextSeq™ 500 High Output Kit for the generation of 80 base pair paired end reads (150 cycles) (Illumina, cat # FC-404-1002).
GEO Accession
GSM3612621