title: GSM8033691: D2_2; Mus musculus; RNA-Seq
library_construction_protocol: To isolate macrophages, calf muscle from the ischemic limb was collected and digested in 2 mg/ml Collagenase IV (Thermo Fisher Scientific, 17104019) / Dispase II (Sigma-Aldrich, D4693-1G) for 1 h at 37°C. After filtration and washing steps, red blood cells were removed with ACK Lysis buffer (Gibco, A1049201). Total RNA for each sample was extracted using RNeasy micro kit (QIAGEN,  74106). Quantification of total RNA was performed using the Tecan microplate reader (DKSH). Quality control of RNA was performed with the Tapestation system (Agilent). 50-100 ng of RNA was used as an input for the Smart-seq2/3 protocol (Picelli et al, 2013, 2014; Hagemann-Jensen et al, 2020) including cDNA synthesis (quantified with the Qubit system for further steps), pre-amplification, tagmentation, and enrichement step. In short, RNA libraries were prepared by performing reverse transcription and template switching using Maxima H Minus reverse transcriptase (ThermoFisher, #EP0753), a template switch oligo and an oligodT primer to generate full length cDNA. cDNA was amplified using the Kapa Hotstart 2x ReadyMix (Roche Diagnostics, #7958935001). 1-3 ng cDNA were then tagmentated using 1.3 µg Tn5 and amplified using Kapa HiFi plus dNTPs (Roche Diagnostics, #07958846001) and the following PCR settings: 72˚C 5 minutes, 98˚C 30 seconds, 10 cycles of 98˚C for 10 seconds, 63˚C for 30 seconds, 72˚C for 1 minute, hold at 4˚C. Libraries were quantified using the KAPA library quantification kit (Roche Diagnostis, #079602), and sequenced at PE150 on a NovaSeq 6000 at Novogene.