GSM3258793: clone_30; Mus musculus; Bisulfite-Seq
RNA was isolated from cell lines containing the expression libraries with Quiagen RNeasy Mini Kit with on-column DNase digestion and reverse transcribed using Takara PrimeScript RT Reagent Kit (#RR047A). For DNA isolation cell pellet was resuspended in Bradleys-Buffer, 6 ul RNaseA (10mg/ml) was added and samples were incubated for 1h at 37 degree C. Subsequently 30 ul protease K was added and samples were incubated at 50 degree C over night. Then DNA was extracted using Phenol and Chloroform. Bio-ChIP was performed as previously described (Baubec et al, 2013). For amplicon Bis-seq, cells containing integrated mutant CGI promoter libraries were thawed, plated at low density and 96 clones picked and expanded to a minimum cell number of 20,000 cells. Cells were lysed with Baradley Buffer (10mM Tris-HCl (pH 7.5), 10mM EDTA (pH 8.0), 0.5% SDS, 10mM NaCl) and DNA extracted with beads (Homemade). DNA was Bisulfite converted using Zymo lightning conversion kit (D5046) and cleaned up using MagBeads (Zymno). Bis-converted DNA was amplified with primers RSG353 and RSG354 (Table 1). Amplified DNA was purified with Ampure beads and sequencing libraries were prepared using the Illumina NEBNext ChIP-seq library prep kit with 96 dual indexing. DNA and cDNA barcodes were amplified with KAPA HIFI Hotstart using Primer DH.P6 and indexing primer (Table 1). PCR products were purified using AmPure XP beads (Beckman Coulter, #A63880) and sequenced using 50 cycle Kit on HiSeq 2500.