GSM9892432: Hippocampus, total RNA-Seq, 1186; Homo sapiens; ncRNA-Seq
We used human brain samples with the age range of 54-110 years old. Frozen hippocampal tissues were collected from a coronal section of the caudal hippocampus at the level of the lateral geniculate nucleus at the Boston University Alzheimer's disease Research Center (BU-ADRC). After RNA extraction, RNA was purified using RNeasy Mini Kits (Qiagen), and the RNA integrity number was calculated using an RNA 6000 Pico assay on a Bioanalyzer 2100 (Agilent Technologies). Total RNA Seq Library Prep:  Using the Kapa RNA HyperPrep Kit with RiboErase (KR1351), rRNA is depleted starting from 25-1000ng of total RNA by hybridization of rRNA to complementary DNA oligonucleotides, followed by treatment with RNase H and DNase to remove rRNA duplexed to DNA.  Samples are then fragmented using heat and magnesium.  1st strand synthesis is performed using random priming.  2nd strand synthesis incorporates dUTPs into the 2nd strand cDNA.  Adapters are then ligated and the library is amplified.  Strands marked with dUTPs are not amplified, allowing for strand-specific sequencing.  Indexed libraries that meet appropriate cut-offs for both quantity and quality are quantified by qRT-PCR using a commercially available kit (KAPA Biosystems) and insert size distribution determined with the LabChip GX or Agilent Bioanalyzer. Samples with a yield of ≥0.5 ng/ul are used for sequencing. All libraries were prepared in a strand-specific manner. RNA Seq Library Prep for PolyA+ and PolyA- sequencing: Using the Kapa RNA HyperPrep Kit with RiboErase, rRNA is depleted starting from 500ng of total RNA by hybridization of rRNA to complementary DNA oligonucleotides, followed by treatment with RNase H and DNase to remove rRNA duplexed to DNA. Following rRNA depletion, mRNA is selected by oligo-dT beads using the KAPA mRNA HyperPrep kit. After the first and second capture, the PolyA minus fraction is kept. After the PolyA captures, the minus fractions are concentrated using the Zymo RNA Clean and Concentrator Kit. Both the PolyA plus and minus fractions are fragmented using heat and magnesium. Following first-strand synthesis with random primers, second-strand synthesis, and A-tailing are performed with dUTP for generating strand-specific sequencing libraries. Adapter ligation with 3' dTMP overhangs are ligated to library insert fragments.  Library amplification amplifies fragments carrying the appropriate adapter sequences at both ends.  Strands marked with dUTP are not amplified. Indexed libraries that meet appropriate cut-offs for both quantity and quality are quantified by qRT-PCR using a commercially available kit (KAPA Biosystems) and insert size distribution determined with the Agilent Tapestation.