title: NextSeq 500 paired end sequencing; ATAC-seq of in vitro reprogrammed mouse pericytes to oligodendrocytes
design: ATAC-seq of in vitro reprogrammed mouse pericytes to oligodendrocytes
library_construction_protocol: Pericytes (PCs) were cultured in pericyte medium. Mouse embryonic fibroblasts (MEFs) were cultured in MEF medium. Oligodendrocytes (pOPCs and iOPCs) were cultured in OPC medium. The cells were cultured in a humidified incubator at 37C under 5% CO2.  To induce differentiation of MEFs or PCs into oligodendrocytes, PC and MEFs were transduced with virues containing Olig2/Sox10 (2F-pOPCs) or Olig2/Sox10/Nkx6.2 (3F-pOPCs). 20.000 cells were harvested and washed once in cold PBS for 5 min at 500 x g at 4 C. The pellet was gently resuspended in 50ul of cold lysis buffer and spun down immediately at 500 x g for 10 min at 4 C. Transposition reaction mix with the enzyme and transposition buffer from Illumina Nextera DNA Library Preparation Kit was added to the pellet and incubated for 30 min at 37 C. The DNA was purified after transposition using a Qiagen MinElute kit and eluted in 10ul of the provided elution buffer. Eluted after transposition DNA was PCR amplified for a total of 11-13 cycles using barcoded primers from Illumina Nextera XT Index Kit v2. DNA was purified and adapters were removed using Ampure beads (1.4:1.0 beads:sample ratio). The quality and concentration of the eluted libraries were determined using an Agilent Bioanalyzer HS chip and a Qubit fluorometer. Libraries were sequenced on Illumina NextSeq 500 in paired-end mode.