GSM4194807: 24-month_APP23_1; Mus musculus; RNA-Seq
The hippocampus was first lysed and homogenized in EZ lysis buffer(Sigma, NUC101). with 0.4 U/μL RNasin Plus Ribonuclease inhibitor (Promega, N2615) and 0.2 U/μL SUPERase In RNase inhibitor (Life Technologies, AM2696) in a 7 mL Dounce tissue grinder to release the nuclei. The homogenized tissue was then filtered through a 40 μm cell strainer, washed and blocked with staining buffer (PBS containing 0.5% BSA and 0.2 U/μL RNasin Plus Ribonuclease inhibitor (Promega, N2615)) then stained using Rabbit recombinant monoclonal NeuN antibody (Abcam, EPR12763, 1:2000) and DAPI (Life Technologies) for neuronal nuclei and all nuclei, respectively. Both NeuN antibody and DAPI were diluted in staining buffer. The nuclei were then washed with staining buffer, re-suspended in 1 ml staining buffer, and proceeded directly to FACS sorting. Single nuclei were vortexed for 90 s and incubated at 72℃ for 3 min to release all RNAs. The mRNAs were captured with barcoded reverse transcription primers with oligo-dT and UMIs and reverse-transcribed into first-strand cDNAs. The cDNAs were amplified using PCR, and products with different barcodes were pooled together. The index sequence with biotin was added to the 3' ends of the pooled cDNA using PCR amplification. Following cDNA fragmentation using Covaris S220 Focused-ultrasonicator, Dynabeads MyOne Streptavidin C1 (Invitrogen, 65002) was used to capture the 3' cDNAs and the sequencing libraries were constructed with Kapa Hyper Prep Kit (Kapa Biosystems, KK8505).
GEO Accession
GSM4194807