nuclearRNAseq_sham_operated3
Rattus norvegicus
source_name
sham-operated 3
cell type
PCM1-positive nuclei from left ventricle of the heart
strain
Sprague-Dawley
chip antibody
NA
hypertrophy induction
Male Sprague Dawley rats of ~170 g were anesthetized and ventilated through endotracheal intubation with a mixture of 69% N2O, 29% O2 and 2% isoflurane. Sham-operated animals underwent the same surgical procedure as those receiving aortic banding, except tightening the silk suture around the ascending aorta proximal to the brachiocephalic trunk. Buprenorphine was given as postoperative analgesia.
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.