title: GSM4503985: Methylseq Normal pancreas -2; Mus musculus; OTHER
library_construction_protocol: RNAeasy plus Mini or Micro Kit (QIAGEN) for RNA and PureLink Genomic DNA mini kit (invitrogen) for genomic DNA. For RNA-seq library, RNA (in vitro and in vivo samples; 200 ng, FACS-sorted mCherry-positive tumor cells; 50 ng) was prepared for library construction. RNA-seq libraries were generated using the TruSeq Stranded mRNA LT sample prep kit (Illumina) or NEBNext Ultra II Directional RNA Library Prep kit for Illumina (NEB). The libraries were sequenced on a NextSeq500 (Illumina) as 86 bp single reads. For whole genome bisulfite sequencing library, 500ng of DNA was fragmented by sonication (Covaris) and ligated with methylated adapters using TruSeq™ Nano DNA Library Prep Kit and TruSeq™ ChIP Libary Preparation Kit (Illumina). Subsequently, DNA was bisulfite-treated using EZ DNA Methylation-Gold Kit™ (ZYMO RESEARCH). Final library amplification was performed using Pfu Turbo Cx (Agilent Technologies). For methyl-seq library, 1μg of DNA was fragmented by sonication. Subsequently, library preparation was performed using the SureSelectXT Mouse Methyl-Seq Reagent Kit (Agilent Technologies). DNA was bisulfite-treated using the EZ DNA Methylation-Gold Kit™. The libraries were then sequenced on a Hiseq 2500 (Illumina) in a rapid mode as paired-end reads (Read1:109 bp, Read2:108bp). For ATAC-seq library, cells were lysed with a lysis buffer (10mM Tris-HCl pH7.4, 3mM MgCl2, 10mM NaCl and 0.1% IGEPAL CA-630). Yielded nuclei were suspended in the transposase reaction mix (25 µl of 2X TD Buffer (Illumina), 2.5 µl of TD Enzyme 1 (Illumina), and 22.5 µl of nuclease-free water), and incubated for 30 minutes at 37 ℃. Fragmented DNA was then purified using MinElute PCR purification kit (QIAGEN). After that, fragmented DNA was amplified by PCR to obtain ATAC-seq libraries. The libraries were paired-end sequenced with a length of 50 bp on Hiseq 1500 (Illumina).
attributes.tag: GEO Accession
attributes.value: GSM4503985