title: NextSeq 500 paired end sequencing; Genome-wide identification and DNA methylation of human full-length LINE-1 (L1) retrotransposons in HCT116 cells treated with 5-aza-2'-deoxycytidine.
design: Genome-wide identification and DNA methylation of human full-length LINE-1 (L1) retrotransposons in HCT116 cells treated with 5-aza-2'-deoxycytidine.
library_construction_protocol: HCT-116 cells were cultured in McCoy medium supplemented with 10% FBS, 100 U/mL penicillin and 100 µg/mL streptomycin. For 5-aza-2'-deoxycytidine (DAC) treatment, cells were plated at a density of 100,000 cells/well in 6-well plates and treated with DAC at a final concentration of 1 μM for a total of 5 days. Fresh medium and drug were added daily for the first 3 days. In control samples (mock), 5-aza was replaced by DMSO. Genomic DNA was extracted and prepared using QiaAmp DNA Blood mini kit (Qiagen) In brief, bs-ATLAS-seq follows these steps: fragmentation of genomic DNA by sonication, ligation of a methylated adapter, bisulfite treatment of linker-ligated DNA, suppression PCR-amplification of L1 5' junctions, and asymmetric Illumina paired-end sequencing (90 bp + 210 bp). Read #1 starts from a random position (sonicated DNA break), while read #2 starts from a fixed sequence in L1 5' UTR (internal promoter). With this design, read #2 covers the 15 most 5' CpG sites (1-207, relative to L1HS consensus sequence). Read #1 has the same orientation as the L1 element.