GSM9110331: CD81,HEK293T,pTET,Dox,72hrs,rep1; Homo sapiens; OTHER
Genomic  DNA  was  extracted  from  cells  using  the  Agencourt  DNAdvance  Kit  (Beckman  Coulter). PCR primers  flanking  the  target  site  were  designed  to  amplify  200-300bp  amplicon  using  a  2  stage  PCR method.   Primer sequences for amplicon sequencing are provided in Table 5. The PCR includes genomic DNA,  i5  and  i7  indexed  adapters,  target specific primers with universal adapters, and PrimeTime Gene Expression  Master  Mix  (IDT).   The  first stage of PCR amplifies the target specific amplicons with target specific  primers  flanked  by  universal  adapters  at  60°C  Tm  for  30  cycles.  The  second  stage  of  PCR incorporates the indexed sequencing adapters in the target specific amplicons at  55°C for 4 cycles.  We use  dual  end  indexing  by  incorporating  i5  and  i7  10bp  indexes  by  IDT  to  uniquely  label  amplicons produced  from  each  PCR.   The  PCRs  are  assembled  using Echo 525 Acoustic Liquid Handler (Beckman Coulter).  Following  PCR,  the  amplicons  from  all  of  the  reactions  are  pooled  together.   The  amplicon library  pool  is  cleaned  twice  using  KAPA  Pure  Beads(Kapa)  at  1:1 ratio to remove unused primers and index  adapters.   The  quality  and  quantity  of  the  clean amplicon library pool is determined using 4200 TapeStation  System  (Agillent)  and High Sensitivity D1000 DNA ScreenTape. The amplicon library pool is diluted  to  15pM  with  5%  PhiX as per Illumina MiSeq library preparation procedure, and sequenced on Illumina MiSeq, dual read, 150/150 cycles to produce 300bp sequences using the Miseq V2 kit (Illumina). Primer sequences used for amplicon sequencing are listed in Supplementary Table 5.