title: GSM6870679: Microglia HIV-1 (WT) CTCF-KD rep1; Homo sapiens; OTHER
library_construction_protocol: Infected C20 microglia cells were harvested 3 d.p.i and DNA was isolated and sonicated. The LM PCR protocol was adapted from (Serrao et al. 2016). Sonicated gDNA ends were repaired using End-It™ DNA End-Repair Kit (Epicentre) and A-tailing was performed using NEBNext® dA-Tailing Module (NEB). An asymmetric double stranded linker was annealed overnight at 12°C (800 U T4 ligase, 10x ligase buffer, 1.5 mM linker). After purification, first nested PCR with linker and LTR specific primers was performed. The second nested PCR with the same linker specific primer and an inner LTR specific primer containing Illumina sequencing index was performed. Library of viral-genome junctions was constructed by PCR with in-house primers annealing to HIV-1 LTR region and linker region, containing Illumina adapters and dual indexes.