2.5. Additional Materials for NGS of sgRNA or Barcodes

• PCR and NGS primers for sgRNA/barcode amplification and sequencing (IDT): See NGS Cassette Diagram, provided as a link or attachment in email upon shipment of product or in Product Certificate of Library NGS Prep Kit.

For your convenience, we also offer Library NGS Prep Kits which provide primers and reagents to prepare samples screened with your library. The NGS Prep Kit and Supplementary Primer Set each contain 12 unique indexing primers that can be used to multiplex up to 24 screening samples on a flow cell or lane.

• Titanium Taq DNA Polymerase with PCR buffer (Clontech-Takara, Cat.# 639242)
• dNTP Mix (10 mM each) (Thermo Fisher, Cat.# 10297018)
• QIAquick PCR Purification Kit (QIAGEN, Cat.# 28106)
• QIAquick Gel Extraction Kit (QIAGEN, Cat.# 28706)
• PhiX Control (Illumina, Cat.# FC-110-3001)
• NGS Reagent Kits, for NextSeq and HiSeq (Illumina):
  

  Platform	Kit Type	Illumina Cat.#	Description
  NextSeq® Single Read (SR) or Paired End (PE)	Sequencing & Cluster Gen.	20024906	NextSeq 500 v2.5 Kit (75 cycles, High-Output)
  HiSeq® 2000/2500 Single Read (SR)	Sequencing	GD-401-3001	TruSeq SBS Kit v3 – HS (50 cycles)
  	Cluster Gen.	FC-401-3002	TruSeq SR Cluster Kit v3-cBot-HS
  HiSeq® 2000/2500 Paired End (PE)	Sequencing	GD-401-3001	TruSeq SBS Kit v3 – HS (50 cycles)
  	Cluster Gen.	PE-401-3001	TruSeq PE Cluster Kit v3-cBot-HS
  HiSeq® 3000/4000 Single Read (SR)	Sequencing	FC-410-1001	HiSeq 3000/4000 SBS Kit (50 cycles)
  	Cluster Gen.	GD-410-1001	HiSeq 3000/4000 SR Cluster Kit
  HiSeq® 3000/4000 Paired End (PE)	Sequencing	FC-410-1001	HiSeq 3000/4000 SBS Kit (50 cycles)
  	Cluster Gen.	PE-410-1001	HiSeq 3000/4000 PE Cluster Kit

NOTE: See Illumina website for information on kits for other Illumina instruments (e.g. NovaSeq, NextSeq 1000/2000, MiSeq).

Other than the specific reagents and instruments listed above, the protocols assume the user has access to standard materials (e.g., polypropylene tubes, pipette tips), equipment (table top centrifuges, pipettes, scales), and common reagents (e.g., TE buffer, ethanol) and buffers used in a typical life science laboratory.