title: GSM4602044: G_4 [AVN-2]; Mus musculus; RNA-Seq
library_construction_protocol: Whole mouse hearts were harvested at P28. Pure CM Nuclei were isolated using 2.2M sucrose cushion for all samples. MAN-IP was performed for 3 CCS compartment specific Cre driver lines. For the 3 Cre driver lines, microdissection was performed to enrich for tissue of interest and obtain specific cell-type nuclei (SAN, AVN, and VCS). CCS specific nuclei were pooled from multiple hearts to ensure enough starting nuclear RNA for good quality library preparation The nuclei suspension (CM nuclei from whole heart, CCS enriched nuclei, etc.) was mixed with equal volume of DNA/RNA Shield (2X concentrate provided in ZR-Duet DNA/RNA MiniPrep Plus kit, #D7003), and RNA was extracted using the same kit following the instruction manual. Qubit quantification was used to estimate RNA concentration. Next, RNA was concentrated to 10µl volume using the RNA Clean and Concentrator-5 Kit (Zymo Research, #R1015). RNA sequencing libraries were made using the TruSeq Stranded Total RNA with Ribo-Zero Human/Mouse/Rat kit (Illumina) according to manufacturer instructions. The resulting RNAseq libraries' quality and concentration were assessed using a 2100 Bioanalyzer with the High Sensitivity DNA Kit (Agilent) and a Qubit Fluorometer with the Qubit dsDNA HS Assay Kit (Life Technologies). RNA-seq libraries were generated from ribosomal depleted RNA and were sequenced on a HiSeq 2500 (50 bp, Illumina).
attributes.tag: GEO Accession
attributes.value: GSM4602044