title: GSM8074176: Right ventricle, no treatment, replicate 4; Mus musculus; RNA-Seq
library_construction_protocol: Following excision of the mouse heart, the atria and atrioventricular valves were surgically removed. We then dissected the left and right ventricle free walls, and washed the tissue in chilled DPBS. Remaining septal tissue was discarded. LV and RV tissue samples were then minced and digested in a solution of 2 mg/ml Collagenase type II (Worthington Cat#: LS004176) and 2 mg/ml Dispase II (Sigma Cat#: D4693) at 37°C for 45 minutes. Trituration was performed with a 10mL serological pipette at the 15, 30, and with a 1 mL pipette at the 45 minute mark. Samples were then filtered through 70 µm strainers into 30mL of chilled DPBS, split into two 15mL Eppendorf tubes, and centrifuged for 7 min at 500g. . After decanting, pellets were resuspended in 100uL of Hank's Balanced Salt Solution (HBSS containing 2% FBS, and incubated with CD31 and CD45 magnetic beads for 15 minutes. Endothelial (CD31+) and hematopoietic cells (CD45+) were subsequently removed using the AutoMacs instrument (Miltenyi Biotec). The remaining cells were stained with DAPI, and live CFs (GFP+, DAPINeg) were sorted on the Aria IIu (BD Biosciences) into HBSS containing 2% FBS. RNA was then isolated using the RNeasy Micro Kit (Qiagen, Cat.#: 74004) according to manufacturer's instructions and stored at -80°C until library construction. RNA samples were assessed on a RNA 6000 Pico chip (Agilent Technologies) to determine sample quality. Seven nanograms of total RNA per sample was used to prepare RNA libraries using the SMARTer Stranded Total RNA-seq Kit V3– Pico Input Mammalian (Takara Bio USA Inc.). Briefly, the samples underwent first-strand synthesis via random priming oligos on the 3' end. A template switching oligo mix (TSO) is then incorporated to allow the RT reaction to continue replicating the 5' of the RNA strand. Following this, the samples were PCR amplified to incorporate full-length Illumina adapters and sample barcodes by binding to either the TSO stretch on the 5' end, or the random priming oligo sequence on the 3' end. The amplified cDNA is treated with ZapR which specifically targets ribosomal RNA in the presence of mammalian-specific R-Probes. This process leaves non-ribosomal RNA untouched, while ribosomal RNA are cleaved, leaving them non-amplifiable. A final PCR reaction is done to enrich the uncut strands of cDNA to generate the final RNA library. Final RNA library sizing was verified on High sensitivity DNA kit (Agilent Technologies) using the Agilent Bioanalyzer while library concentration was quantified by qPCR using the KAPA SYBR FAST qPCR kit (Roche). All libraries were normalized and pooled together, denatured with 0.2N NaOH and diluted to a final concentration of 250 pM.  Pooled libraries were loaded onto an Illumina Novaseq V1.5 cartridge for cluster generation and sequencing on an Illumina Novaseq 6000 instrument (Illumina) Paired-end 101 bp protocol to achieve  ~ 40 million reads per sample.