GSM6879557: P7C3, ApoeKO MifKO, 24 weeks HFD, mouse 159; Mus musculus; RNA-Seq
5 μm OCT-embedded frozen sections from the BCA were gently thawed (room temperature, 5 min). Sections were immersed in 50 μL of freshly prepared RNA isolation buffer (20% proteinase K (Qiagen, Hilden, Germany) and 1:2400000 ERCC RNA spike-in mix in buffer PKD (Thermo Fisher Scientific) for approximately 30 seconds to permit lysis and homogenization of the whole BCA tissue. A 200-uL tip was used to scratch the BCA tissue lysate from the glass slide under a microscope. The BCA lysates from 3-4 BCA sections were pooled and collected in a 1.5 mL reaction tube and frozen at -80°C. The RNA in the BCA lysates was purified and washed with oligo dT25 magnetic beads (Thermo Fisher Scientific), followed by removing the beads via incubating the samples at 80°C and placing the samples on a magnet.  For library construction, 1 ng RNA was taken from each sample for reverse transcription and cDNA amplification following a Smart-seq2 protocol, followed by library construction as described before (34, 35). Briefly, samples were first thawed and then incubated for 3 min at 72°C and immediately placed on ice followed by the steps of reverse transcription (RT) and pre-amplification. The cDNA was then cleaned using AMPure bead (Beckman-Coulter) clean-up at a 0.7:1 beads:PCR product ratio. Libraries were assessed using Bio-analyzer (Agilent, catalog no. 2100), and the High Sensitivity DNA analysis kit, and also fluorometrically using Qubit's DNA HS assay kits and a Qubit 4.0 Fluorometer (Invitrogen, Life Technologies) to measure the cDNA concentrations. After normalization to 160 pg/uL, 1.25 μL of cDNA samples were used for library construction using in-house Tn5 transposase. Libraries were barcoded and pooled, then cleaned up with three rounds of of AMPure bead (Beckman-Coulter) at a 0.8:1 ratio beads:library. Libraries were sequenced 2 x 100 reads bp paired-end on a DNB-seq platform to an average depth of 3 × 106 reads per sample. Sequencing was performed by BGI Tech Solutions (Hongkong) Co., Limited (Hongkong, China).