GSM9615607: RiboTag IP-Camk2(Excitatory) | KO | female | S1 | Sample 83; Mus musculus; RNA-Seq
For each mRNA isolation experiment, mice were anaesthetized with isoflurane, followed by cardiac perfusion with ice cold choline ACSF containing 132 mM choline, 2.5 mM KCl, 1.25 mM NaH2PO4, 25 mM NaHCO3, 7 mM MgCl2, 0.5 mM CaCl2, and 8 mM D-glucose). Coronal sections (1.5 mm thick) containing S1 (A/P -0.5 to +1.82 mm, M/L 3.0 to 4.0 mm) and V1 (A/P +3.5 to 5.5 mm, M/L 1.5 to 3.0 mm) were collected using Stainless-Steel Brain Matrices (Zivic instruments) in chilled choline, from which S1 and V1 were dissected bilaterally. Samples were independently homogenized with a 2 mL Dounce homogenizer in 1 mL of supplemented homogenization buffer containing 1% w/v, HB-S: 1 mM DTT, protease inhibitors (1X), RNAsin (200 units/mL), cycloheximide (2 mg/l), and heparin (1 mg/mL). 100 μL of the homogenate was used for isolating bulk RNA from S1-V1. To isolate mRNA of HA-tagged Pvalb-Cre or Camk2a-Cre from non-HA tagged ribosomes, samples were incubated for 4 h with an anti-HA antibody (1:180, 5 μL in 900 μL of homogenate; 901513 Covance Anti-HA antibody), followed by overnight incubation with magnetic beads (Pierce Protein A/G Magnetic Beads 88802, 200 μL) at 4oC at 15 rpm centrifugation. The supernatant was washed 3 times with 800 μL of high-salt buffer (Tris 50 mM, KCl 300 mM, MgCl2 12 mM, 10% NP-40 (1%), DTT 1 mM, cycloheximide 100 μg/mL, dH2O), centrifuged at 15 rpm for 10 min at 4 oC. For RNA extraction, we used Zymo Research Direct-zol RNA Miniprep kit (R2051) for the homogenate (input), and the Zymo Research Direct-zol RNA Microprep kit (R2061) for the bead-antibody-protein sample (IP); RNA was eluted with DNAase/RNAse-free water. RNA Integrity Number (RIN) scores were used to evaluate the integrity of RNA through the ratio of 28S:18S ribosomal RNA (Agilent TapeStation 4200). We set a minimum RIN score of 7 for our samples. RNA-seq libraries were prepared using Ovation® RNA-Seq System V2. Libraries were multiplexed for paired end 100 bp sequencing on NovaSeq S2, with a read depth of ~45 million reads on average.