title: Illumina HiSeq 4000 paired end sequencing; Effects of Post-Myocardial Infarction Heart Failure on the Bone Vascular Niche (Mouse Data)
design: Effects of Post-Myocardial Infarction Heart Failure on the Bone Vascular Niche (Mouse Data)
library_construction_protocol: Bone marrow aspirates were taken from the femur of adult mice at day 0 (no evidence of myocardial infarction) and at day 7 and day 28 after myocardial infarction. (Associated to E-MTAB-10432: Bone marrow aspirates were obtained from healthy volunteers without any evidence of coronary artery disease in their history (median age 31 years [IQR 27-37]) as well as from patients suffering from post-infarct chronic heart failure (CHF) with severely reduced left ventricular function, undergoing intracoronary infusion of bone marrow mononuclear cells within the REPEAT trial (Repetitive Progenitor Cell Therapy in Advanced Chronic Heart Failure; NCT 01693042, N=19). All controls and patients provided written informed consent. The ethics review board of the Goethe University in Frankfurt, Germany, approved the protocols (Approval No. 160/15 for healthy controls and the study consent for REPEAT trial)4. The study complies with the Declaration of Helsinki. Patients were eligible for inclusion into the study if they had stable CHF symptoms described by New York Heart Association (NYHA) classification of at least II, had a previous successfully revascularized myocardial infarction at least 3 months before bone marrow aspiration, and had a well-demarcated region of left ventricular dysfunction on echocardiography. Exclusion criteria were the presence of acutely decompensated heart failure with NYHA class IV, an acute ischemic event within 3 months prior to inclusion, a history of severe chronic diseases, documented cancer within the preceding 5 years, or unwillingness to participate.)  Bone RNA isolation was performed with Direct-zol RNA Miniprep Kits from (Zymoresearch, R2051) according to manufacturer protocol. RNA concentration was measured by nanodrop (Thermofisher scientific). For cDNA synthesis, 1µg RNA was reverse-transcribed using M-MLV reverse transcriptase (Invitrogen, 28025-021). Quantitative PCR was performed with TaqMan fast Mastermix (435204, Applied Biosystems) and the following Taqman probes: IL1b (Mm00434228_m1) and P0 (Mm00725448_s1).  Cellular suspensions were loaded on a 10X Chromium Controller (10X Genomics) according to manufacturer's protocol based on the 10X Genomics proprietary technology. Single-cell RNA-Seq libraries were prepared using Chromium Single Cell 3′ Reagent Kit, v2 (human) and v3.1 Next GEM (murine) (10X Genomics) according to manufacturer's protocol. Briefly, the initial step consisted in performing an emulsion capture where individual cells were isolated into droplets together with gel beads coated with unique primers bearing 10X cell barcodes, UMI (unique molecular identifiers) and poly(dT) sequences. Reverse transcription reactions were engaged to generate barcoded full-length cDNA followed by the disruption of emulsions using the recovery agent and cDNA clean up with DynaBeads MyOne Silane Beads (Thermo Fisher Scientific). Bulk cDNA was amplified using a Biometra Thermocycler TProfessional Basic Gradient with 96-Well Sample Block (98°C for 3 minutes; cycled 14×: 98°C for 15s, 67°C for 20s, and 72°C for 1 minute; 72°C for 1 minute; held at 4°C). Amplified cDNA product was cleaned with the SPRIselect Reagent Kit (Beckman Coulter). Indexed sequencing libraries were constructed using the reagents from the Chromium Single Cell 3′ v2 or v3.1 Next GEM Reagent Kit, as follows: fragmentation, end repair and A-tailing; size selection with SPRIselect; adaptor ligation; post-ligation cleanup with SPRIselect; sample index PCR and cleanup with SPRI select beads. Library quantification and quality assessment was performed using Bioanalyzer Agilent 2100 using a High Sensitivity DNA chip (Agilent Genomics).