title: NextSeq 550 paired end sequencing; Iron accumulation drives fibrosis, senescence, and the senescence-associated secretory phenotype
design: Iron accumulation drives fibrosis, senescence, and the senescence-associated secretory phenotype
library_name: Iron_day6_p
library_construction_protocol: To induce pulmonary fibrosis 8-10-week-old C57BL/6 wild-type mice were anesthetized by intraperitoneal injection of a mix containing ketamine (75 mg/kg) and medetomidine (1 mg/kg). Animals were placed on a Tilting WorkStand for rodents (EMC Hallowell) and intubated intratracheally with a 24GA catheter (BD Biosciences), and delivered 30 ul aqueous iron solution containing a concentration of 8,3 mM Fe(II)Sulfate and 8,3 mM Fe(III)Nitrate (in total 16,5 mM iron), control animals received 30 μl phosphate buffered saline (PBS). Iron solution was sterile filtered with a 0.22 µm pore size syringe filter. Mice receiving intratracheal iron developed within a day sepsis-like symptoms, with severe weight-loss, from which day recovered on the fourth day post-iron. We found that treatment with a daily dose of subcutaneous buprenorphine (0.1 mg/kg) considerably mitigated these symptoms, and the overall mortality in this model remained very low (<10%). Mice were sacrificed in CO2 chambers, and lungs were removed, separated into lobes, frozen on dry ice, and stored at -80 °C until the day of nuclei isolation. Nuclei were isolated from the lung lobes using the Chromium 10X nuclei isolation kit following the manufacturer´s instructions. Three single nuclei suspensions were used for transcriptome analysis at IRB Barcelona Functional Genomics Core Facility. Briefly, nuclei isolated using the 10x genomics Chromium Nuclei Isolation with RNase Inhibitor Kit (10x Genomics), were filtered using a 40 μm flowmi cell strainer (SP Bel-Art), and quality controlled on a LUNA-FL automated fluorescence cell counter (Logos biosystems). The suspensions were used to target between 10,000 and 12,000 nuclei per sample. Partition into GEMs was performed using Chip G and the 3' v3.1 chemistry (10X Genomics). Barcoded cDNA was amplified for 13 cycles, quantified, and quality controlled on the Bioanalyzer 2100 using a high sensitivity DNA assay (Agilent). The generated illumina-compatible libraries were quantified using the Qubit dsDNA HS assay (Invitrogen), quality controlled with the Bioanalyzer 2100 DNA HS assay (Agilent) and used to generate an equimolar pool.