GSM4594558: RARRXR_SLOTv2_mEND_rep2_DpnI; Mus musculus; OTHER
DPNI: 10ug genomic DNA DPNII: 20ug genomic DNA Library prep was performed by 3 successive PCRs. In the first PCR, the entire DpnI-digested genomic DNA was used in a 400 uL PCR rxn, and the entire DpnII-digested genomic DNA was used in a 800 uL PCR rxn. 13 cycles of PCR1 (Q5 UltraII mastermix, Ta=66, 60 second 72 deg extension per cycle) was performed. PCR 1 was purified using PCR purification in a single column, eluting in 50 uL. PCR2 was performed for 7 cycles (Q5 UltraII mastermix, Ta=66, 60 second 72 deg extension per cycle) using 22.5 uL of PCR1 product in a 100 uL reaction. PCR 2 was purified in a single column, eluting in 50 uL.  qPCR (Q5 UltraII mastermix with EvaGreen (Biotium), Ta=66, 60 second 72 deg extension per cycle) was used to determine PCR3 cycle counts. PCR3 was performed by subtracting 2-3 cycles from the Ct and using 2.5 uL of a mix of distinct indexed primers from the NEBNext Dual Index Kit for Illumina (New England Biolabs) using 22.5 uL PCR2 product in a 50 uL reaction volume (Q5 UltraII mastermix with EvaGreen (Biotium), Ta=66, 60 second 72 deg extension per cycle). PCR3 products were PCR-purified and Tapestation (Agilent) was used to quantify and pool samples for NGS.
GEO Accession
GSM4594558