GSM2492314: MI-3-CD11cpos; Mus musculus; RNA-Seq
Hearts were digested in RMPI containing 20mg/ML Liberase and 10U/ml DNase (37oC for 45 minutes). Single cell suspensions were filtered through a 100mM strainer and stained with antibodies to allow cDC1s,cDC2s, moDCs and MFs to be isolated by FACS. RNA concentration and purity were determined spectrophotometrically using the Nanodrop ND-1000 (Nanodrop Technologies) and RNA integrity was assessed using a Bioanalyser 2100 (Agilent). Per sample, an amount of 4 ng of total RNA was used as input for the SMART-Seq v4 Ultra Low Input RNA protocol (version “040215”) from Clontech Laboratories, Inc. Subsequently, 1ng of purified cDNA was sheared to 300bp using the Covaris M220. From the sheared material, sequencing libraries were prepared with the NEBNext Ultra DNA Library Prep Kit for Illumina (version 3.0 -8/15), according to the manufacturer’s protocol including a size selection to 250bp insert size with the following minor modifications: only half of the library is subjected to a 15 cycle PCR in the final library amplification step. Sequence-libraries of each sample were equimolarly pooled and sequenced on 2 NextSeq500 flow-cells at 1x75 bp with a v2 high-output kit.