Illumina MiSeq paired end sequencing; SAMHD1 restrains aberrant nucleotide insertions at repair junctions generated by DNA end joining
SAMHD1 restrains aberrant nucleotide insertions at repair junctions generated by DNA end joining
HEK 293 or HeLa cells were seeded and transfected with the indicated constructs for indicated time points.   Sequences for substrates #1-9. Index ACAGTG GCA AAT GGC ATT CTG ACA TCC for -SAMHD1 Exp1, CcAAAG GCA AAT GGC ATT CTG ACA TCC for -SAMHD1 Exp2, GACGAC GCA AAT GGC ATT CTG ACA TCC for -SAMHD1 Exp3, TAATCG GCA AAT GGC ATT CTG ACA TCC for +SAMHD1 Exp1, ACTGAT GCA AAT GGC ATT CTG ACA TCC for +SAMHD1 Exp2, CAACTA GCA AAT GGC ATT CTG ACA TCC for +SAMHD1 Exp3. Sequences for substrates #11-18. Index GCA AAT GGC ATT CTG ACA TCC for +SAMHD1 Exp1, GCA AAT GGC ATT CTG ACA TCC for +SAMHD1 Exp2, GCA AAT GGC ATT CTG ACA TCC for -SAMHD1 Exp3, GCA AAT GGC ATT CTG ACA TCC for -SAMHD1 Exp4. Genomic DNA was isolated using the DNeasy Blood & Tissue Kit (QIAGEN) according to the manufacturers instructions.  Repaired plasmids and genomic junctions were PCR-amplified using specific primers with different 6bp 5â€™ tags. For genomic amplicons, the Nextera XT Index Kit (Illumina) was used. Amplicons were pooled and sequenced on the Illumina MiSeq platform.