GSM8460007: clone Sox2P-reporter 1-44_34 Rv; Mus musculus; OTHER
Sorted pools and clones were expanded and crude lysates were obtained from a full 96 well plate by lysing with 50 µL DirectPCR Lysis Reagent (102-T, Viagen Biotec) supplemented with 100 µg/mL proteinase K, and incubating 2.5 h at 55 °C and 45 min at 85 °C. For unsorted control cell pools, gDNA was extracted after expansion using the ISOLATE II Genomic DNA Kit (BIO-52067, Bioline). SBt integrations were mapped by amplifying and sequencing the ITR-genome junction using a tagmentation-based approach. The procedure was similar to {Schep, 2021, 33848455}, but using the following SB-ITR specific primers: MEP009 (5′ ITR, reverse) or LD027 (3′ ITR, forward) for the linear enrichment and MEP011 (5′ ITR, reverse) or MEP034 (3′ ITR, forward) for PCR.Input for one tagmentation reaction was either 100ng gDNA or 3 µL crude lysate. The 72 °C extension times of all PCRs were increased to 1 min. In addition, the composition of the linear enrichment PCR was changed to 10 μL of tagmented DNA, 2 μL of 1 μM primer, 4 μL dNTPs (10mM), 8 μL 5x Phusion® HF Buffer (NEB), 0.5 μL Phusion® HS Flex polymerase (2 U/μL - NEB), in a final volume of 40 μL. For all clones and some experiments with sorted pools (proof-of-principle experiment, third replicate of the reporter hopping) we halved this reaction to a final volume of 20 µL. For the unsorted control pools, 10 separate tagmentation reactions and library preparations were performed, usually using one set of sequencing indices. Libraries were sequenced with 150 bp paired-end reads using either an Illumina MiSeq system (reagent kit v2) or  Illumina NextSeq 550 system (mid-output kit) including 10% of PhiX spike-in.  Primers are described in the corresponding manuscript.