GSM8215115: 16-319_Superficial,TS,Batch_1; Mus musculus; RNA-Seq
Brain tissues were accessed from Ts2 and normal disomic female mice. Mice were transcardially perfused with ice-cold 0.15 M phosphate buffer as previously described. Brains were immediately flash frozen and 20 μm-thick tissue sections were cryostat cut (−25°C) in the coronal plane and mounted on polyethylene naphthalate (PEN) membrane slides. Slides were kept under desiccant at -80°C until used for immunohistochemistry. RNase-free precautions were employed, and solutions were made with 18.2 mega Ohm RNase-free water. RNA from Batch 1 and Batch 2 for each CA1 subregion 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. LCM was performed on tissue to isolate the entire rostral CA1 pyramidal neuron layer from 3-6 sections (CA1A, ~500-600 pyramidal neurons) and compared that to the pyramidal neurons from the distal deep layer CA1 region(CA1D; 6-12 sections; ~500-1500 cells) and to the distal superficial layer CA1 region (CA1D; 6-12 sections; ~500-1500 cells).  Deep and superficial neurons were selected from the same tissue sections.  Batch 2 collected CA1A, CA1D and CA1S from adjacent sections within each slide (2-4 slides per brain to collect at least ~500 neurons per region), whereas batch 1 was collected with CA1D and CA1S in adjacent slides to the CA1A slide. The SMARTer Stranded Total RNA-Seq kit-Pico input Mammalian (Takara Bio, Mountain View, CA) was employed with minor modifications to normalize neuronal numbers for each sample. RNA-seq samples were quantified (Agilent 2100 HS DNA kit), with all samples reading a minimum of 2nM of the library for QC. For each sample run, library samples were pooled in equimolar concentrations and assayed on an Illumina HiSeq-4000 using a single read 50-cycle protocol at NYUGSOM Genome Technology Center.  Samples were run in multiple batches utilizing the same protocol for each batch.