nuclearRNAseq_control1
Rattus norvegicus
source_name
control 1
cell type
PCM1-positive nuclei from left ventricle of the heart
strain
Sprague-Dawley
chip antibody
NA
hypertrophy induction
Male Sprague Dawley rats of ~280 g not assigned to the exercise protocol were used as control.
isolation of material from sorted nuclei
A solution of at least 100000 sorted nuclei was mixed with 3× its volume of TRIzol LS reagent (Life Technologies) and 2 µL glycogen (Life Technologies), and stored for at most one month at -80°C until further processing according to manufacturer's instructions. Genomic DNA was removed using the TURBO DNA-free kit (Life Technologies) as per the kit’s instructions. RNA was subsequently reverse transcribed using SuperScript III Reverse Transcriptase (Life Technologies) as follows: 3 µg Random Primers (mostly hexamers; Life Technologies) were added to 10 µL RNA and 8 µL 5× First-Strand Buffer. This mixture was heated to 94 °C for 3 minutes for RNA fragmentation, and incubated on ice for at least 1 minute. Next 2 µL dNTP Mix (10 mM) (Life Technologies) were added, as well as 4 µl DTT (100 mM), 40U SUPERase.In, 400U SuperScript™ III Reverse Transcriptase and 8 µg Actinomycin D. Water was added to make up the volume to 40 µL, and the sample was incubated at 25 °C for 5 min 50 °C for 30 minutes and inactivated at 70 °C for 15 min. cDNA-RNA duplexes were purified using 72 µL Agencourt AMPure XP (Beckman Coulter) and eluted in 20 µL of the following second-strand synthesis reaction mix: 0.33 mM dATP, dCTP and dGTP (Life Technologies), 0.33 mM dUTP (Sigma Aldrich), 0.25 U Ribonuclease H (Life Technologies) and 10 U DNA polymerase I (New England Biolabs). The reaction was allowed to proceed for 2.5 hours at 16°C.