GSM8647751: Shuffle_clone_A6; Mus musculus; OTHER
Qiagen DNA mini kit We employed two different strategies for amplicon-seq library construction: 2-primer  and 4-primer. The major difference between these two strategies is that either 2 or 4 primers corresponding to the capture sequences were used to generate the PCR product. In the case of the 2-primer strategy, the P5 Illumina sequencing adapter can only come from the primer that binds CS2 and the P7 adapter can only come from the primer that binds CS1. This precludes identification of recombined shuffle cassettes that contain the same capture sequence on both sides of the loxPsym site. In the 4 primer strategy, primers with P5 and P7 adapters that bind to both CS2 and CS1 are included in the PCR. Theoretically, this would enable us to detect recombined shuffle cassettes with the same capture-sequence on both sides. Two rounds of PCR were performed on genomic DNA extracted from indicated samples to construct sequencing libraries. For the 2-primer experiments, we used 250 ng of genomic DNA prepared using the Qiagen DNeasy kit as input. PCR1 (UMI addition) was performed in a 50 μl reaction with Q5 Polymerase (NEB M0492S), with 2.5 μL of each 10 μM primer (oSP1008 and oSP997) with the following cycling parameters: 98°C - 5 min; 4 cycles of 98°C - 20s, 62°C - 20s, 72°C - 30s; 72°C - 60s. PCRs were cleaned up using AmpureXP beads (1X) and eluted in 11 μl of H2O. PCR2 (sample indexing and sequencing adapter addition) was again performed with Q5 Polymerase in a 50 μl reaction using 10 μl of eluate from the previous step as template and 2.5 μL of each 10 μM sample indexing primer. Progression of PCR2 was monitored in real-time using SYBR green dye and reactions were stopped before saturation (usually 15-18 cycles). Cycling conditions for PCR2:  98°C - 5 min; 15 cycles of 98°C - 10s, 65°C - 10s, 72°C - 20s. Reactions were cleaned up with AmpureXP beads (1X) and eluted in 12 μl of H2O. Sample quality was confirmed on a TapeStation D1000 ScreenTape (Agilent), after which reactions were pooled. Sequencing was performed on an Illumina NextSeq2000 100 cycle kit with the following read lengths: 69 read1, 6 index1, 10 index2 and 53 on read2. For the 4-primer experiments involving loxPsym+ mESCs, 100 ng of genomic DNA prepared using the Qiagen DNeasy kit as used as input. For the 4-primer experiments involving loxPsym+ K562, attB/P+ mESC and attB/P+ K562, 250ng of genomic DNA prepared using the Qiagen DNeasy kit was used as input. PCR1 was performed with a mix of 4 primers (oSP1008, oSP997, oSP1021 and oSP1022) with 0.125 μl of each primer at 100 μM per 50 μl reaction. The rest of the protocol was identical to the 2-primer workflow described above. Primer sequences: oSP1008_996_CS2_Nextera1_10N_F TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNNNNNN CCTTAGCCGCTAATAGGTGAGC oSP1021_1014_CS1_Nextera1_10N_F TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNNNNNN TTGCTAGGACCGGCCTTAAAGC oSP997_CS1_TruSeq2_R GTGACTGGAGTTCAGACGTGTGCTCTTCCGATC TTGCTAGGACCGGCCTTAAAGC oSP1022_CS2_TruSeq_R GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT CCTTAGCCGCTAATAGGTGAGC