Illumina MiSeq paired end sequencing; Analysis of gene deletion induced changes in the transcriptome of human macrophage-like cells
Analysis of gene deletion induced changes in the transcriptome of human macrophage-like cells
Adherent cells were washed and scraped after addition of the Qiagen RLT buffer with βME. Lysis was completed by 15sec vortexing of collected suspensions sgRNA transduced THP-1 cells were maintained in RPMI 1640 (A10491-01, GIBCO) medium supplemented with 10% FBS, 100 U/ml penicillin, 0.1 mg/ml streptomycin and 0.05 mM βME at 37°C in a humidified atmosphere with 5% CO2 . Monocytes were differentiated into macrophages using 50 ng/ml PMA for 48 hours and then rested in PMA-free medium for an additional 24 hours before use Total cellular RNA was isolated by RNeasy Mini kit (74106) with DNAse I digestion (79254) using QIAcube instrument (all Qiagen) according to the manufacturer's instructions. Total cellular RNA was isolated by RNeasy Mini kit (74106) with DNAse I digestion (79254) using QIAcube instrument (all Qiagen) according to the manufacturer's instructions. mRNA was enriched using oligo(dT) beads and  randomly fragmented by adding fragmentation buffer. First cDNA strand  was synthesized using random hexamers primer, after which a custom second-strand synthesis buffer (Illumina) , dNTPs, RNase H and DNA polymerase I were added to initiate the second-strand synthesis. After a series of terminal repair, A ligation and sequencing adaptor ligation, the final double-stranded cDNA library was generated by size selection and PCR enrichment.
Experimental Factor: phenotype
CD5L knockout