GSM8965095: 15-232, 2N, ChN, Batch 3, Medial Septal Neurons; Mus musculus; RNA-Seq
A rapid staining protocol utilizing antibody against choline acetyltransferase (ChAT) (AB144P, Millipore, Burlington MA) was performed to detect cholinergic positive neurons while preserving intact RNA in the unfixed tissue. ChAT-immunoreactive (ir) neurons were identified within the medial septal (MS) region and isolated by LCM (LMD7000)RNA from ~500 MS neukrons was purified using miRNeasy Micro kit (Qiagen) according to manufacturers' specifications. A DNase digestion was performed twice sequentially before the final washes and RNA purification. . Approximately 500 ChAT-ir neurons were isolated per brain before proceeding to RNA isolation and RNA-seq library preparation. The SMARTer Stranded Total RNA-Seq kit-Pico input Mammalian (v1, Takara Bio, Mountain View, CA) was employed with minor modifications during step A to utilize full volume of isolated RNA. Samples were quantified (Agilent 2100 HS DNA kit), with samples showing no peak between 300-500bp re-isolated by LCM, with RNA isolation and library preparation repeated.  Samples were isolated and processed in three independent batches, batch 1 (2N, n=3; 2N+, n=5; Ts n=4, Ts+ n=5), batch 2 (2N, n=2; 2N+, n=2; Ts n=4, Ts+ n=2), batch 3 (2N, n=6; 2N+, n=5; Ts n=3, Ts+ n=3).  For each sample run (Batch 1, 2 and 3), library samples were pooled in equimolar concentrations and assayed on an HiSeq-4000 (Illumina, San Diego, CA) using a single read 50 cycle protocol at NYUGSOM Genome Technology Center (GTC).