M mlv reverse transcription reactions

Total RNA was extracted from cells using RNeasy kit (Qiagen) with on column DNAse I digestion. Next 1 ug of RNA was used for M-MLV reverse transcription reactions (Invitrogen). cDNA was diluted and quantified using TaqMan qPCR specific for HIV-1 Gag and Env genes and cellular beta-actin gene as a reference (Table SI.4) under the same protocol like genomic DNA but analyzed using relative quantification mode.

Total RNA was extracted from Jurkat cells using RNeasy kit (Qiagen) with on column DNAse I digestion. Next 0.5 ug of RNA was used for M-MLV reverse transcription reactions (Invitrogen). For gRNA expression screening specific reverse primer (pX260-crRNA-3′/R, Table I.3 in S1 Experimental Procedures) was used in RT reaction followed by standard PCR using target A or B sense oligos as a forward primers (Table I.5 in S1 Experimental Procedures) and agarose gel electrophoresis. For checking neighboring genes expression oligo-dT primer mix was used in RT and cDNA was subjected to SybrGreen real time PCR (Roche) using mRNA specific primer pairs and β-actin as a reference (Table I in S1 Experimental Procedures).