GSM5710373: RNA glia_f_12_9; Mus musculus; RNA-Seq
Arcuate nuclei (ARH) were microdissected from coronal sections.  Nuclei were isolated by ultralcentrifugation, stained with Anti-NeuN (Millipore ABN78, 1:2000) and purified by FACS. DNA extracted by QIAGEN AllPrep Micro kit, RNA by RNA-STAT60 Tagmentation-based whole-genome bisulfite library preparation was performed on genomic DNA extracted from ARH NeuN+ and NeuN- nuclei as previously described (Wang et al., 2013).  Briefly, adaptors were pre-annealed and transposomes were assembled according to the protocol described in (Wang et al., 2013).  Next, 10-30 ng of mouse genomic DNA was tagmented by adding the assembled transposome and incubating at 55C for 8 min. Following purification using SPRI beads, ligonucelotide replacement and gap repair were performed using Ampligase and T4 DNA Polymerase. The product was purified using SRPI beads and followed by bisulfite treatment using the EZ DNA Methylation Gold kit (Zymo Research). Subsequently libraries were generated by PCR amplification using KAPA 2G robust hotstart ready mix. After purification using SPRI beads, libraries were diluted and loaded onto Illumina Hiseq2500 Sequencer for 100 bp paired-end sequencing. Libraries for RNA sequencing were prepared using the SMART-Seq v4 Ultra Low Input kit (Takara Clontech) according to the manufacturer's instructions.  Briefly, purified RNA was incubated with lysis buffer for 5 minutes.  First strand cDNA synthesis was performed using the included 3-SMART-seq CDS primer II and V4 oligonucleotide.  cDNA was amplified using PCR Primer II A, and subsequently purified using Ampure XP beads (Beckman). Illumina libraries were prepared using Nextera XT DNA library preparation kit (Illumina), and sequenced on the Illumina Hiseq 2500 platform generating 100 bp paired-end reads.