title: Illumina HiSeq 2500 sequencing; CRISPR enhancer scan to detect critical elements for the regulation of EVI1 in a translocated enhancer
design: CRISPR enhancer scan to detect critical elements for the regulation of EVI1 in a translocated enhancer
library_construction_protocol: For each replicate, a total of 120 million cells were infected with 3-4% transduction efficiency to ensure that each sgRNA is represented predominantly as a single lentiviral integration in >1000 cells. After neomycin drug selection (1 mg ml−1) for 7 days, D0 samples were obtained (5 million cells per replicate), and cells were subsequently cultured in the presence of 1 µg ml−1 doxycycline (Dox). Culture medium was exchanged every 2 days. After 5 days (d5) and 7 days (d7), about 1 million sgRNA-expressing (GFPlow) cells were sorted for each replicate using a FACS AriaII (BD Biosciences).  The MUTZ3 cell lines were cultured in αMEM (HyClone) with 20% fetal calf serum (FCS) and 20% conditioned 5637 medium. The 293T were cultured in DMEM (Gibco) with 10% FCS. All cell lines were supplemented with 50 U/mL penicillin and 50 μg/mL streptomycin. Viable frozen AML cells were thawed and resuspended in IMDM medium supplemented with: 20% BIT medium (Stem cell technologies), 1x β-mercaptoethanol (1000x Life technologies), 6 µg/ml LDL (Sigma Aldrich), human IL6, IL3, G-CSF, GM-CSF at 20 ng/ml and FLT3, SCF at 50 ng/ml (Peprotech). To design a high-resolution sgRNA library for the enhancer scan, we considered all possible sgRNA target sites containing a canonical Cas9 PAM site (NGG) on both strands of the minimal 18 kb translocated region. sgRNAs containing a G in positions 1-3 of the 20nt target site were trimmed at this position to favor 20-, 19- or 18-mers (in this order of priority) containing a natural G at the 5'end as previously described (Michlits et al., 2020). For all other sgRNAs, a G was added to the 5'end (resulting in a 21-mer). Subsequently, all sgRNAs showing (1) a high number of target sites in the human genome (>5 with no mismatch, or >20 with 1 mismatch), (2) a BsmBI site (interfering with cloning), or (3) a polyA signal (interfering with packaging) were filtered out. In addition, we added a number of negative controls (82 sgRNAs targeting the AAVS1 region) as well as positive controls (5 sgRNAs targeting EVI1 as well as 313 sgRNAs covering 5 kb of the breakpoint in MUTZ3 cells). The final library of 3239 sgRNAs (Table S1) was synthesized with overhangs for PCR amplification and cloning as one oligo pool (Twist Bioscience) and cloned into the lentiviral vector sgETN as previously described (Michlits et al., 2020). The pool of 3239 sgETN-sgRNAs was transduced in triplicate into MUTZ3-EVI1-GFP. For each replicate, a total of 120 million cells were infected with 3-4% transduction efficiency to ensure that each sgRNA is represented predominantly as a single lentiviral integration in >1000 cells. After neomycin drug selection (1 mg ml−1) for 7 days, T0 samples were obtained (5 million cells per replicate), and cells were subsequently cultured in the presence of 1 µg ml−1 doxycycline (Dox). Culture medium was exchanged every 2 days. After 5 days (T5) and 7 days (T7), about 1 million sgRNA-expressing (GFPlow) cells were sorted for each replicate using a FACS AriaII (BD Biosciences). Genomic DNA from T0, T5 and T7 samples was isolated by two rounds of phenol extraction using PhaseLock tubes (5PRIME), followed by isopropanol precipitation. Deep-sequencing libraries were generated by PCR amplification of sgRNA guide strands using primers that tag the product with standard Illumina adapters and a 4 bp sample barcode in a 2 step-PCR protocol. For each sorted sample, all DNA was used as template in multiple parallel 50-μl PCR reactions, each containing 250-500 ng template, 1x AmpliTaq Gold buffer, 0.2 mM of each dNTP, 2 mM MgCl2, 0.3 μM of each primer and 1U AmpliTaq Gold (Invitrogen), which were run using the following cycling parameters: 95 °C for 10 min; 28 cycles of 95 °C for 30 s, 52 °C for 45 s and 72 °C for 30 s; 72 °C for 7 min. PCR products (367 bp) were combined for each sample and Ampure purified. For the T0 samples and a DNA-pool sample the amount of input DNA necessary to get a 1000x coverage was used as input in the PCRs. For the second PCR 10ng of input was used per PCR using the following cycling parameters: 95 °C for 10 min; 8 cycles of 95 °C for 30 s, 57 °C for 45 s and 72 °C for 30 s; 72 °C for 7 min. PCR products (448 bp) were combined for each sample and Ampure-purified. Libraries were sequenced equimolarly on an Illumina HiSeq 2500 (Illumina) by the Next Generation Sequencing Facility at Vienna BioCenter Core Facilities (VBCF), member of the Vienna BioCenter (VBC), Austria. Multiple experiments (different time points and sorted fractions) were sequenced simultaneously, each identified by a unique barcode.  Genomic DNA from D0, D5 and D7 samples was isolated by two rounds of phenol extraction using PhaseLock tubes (5PRIME), followed by isopropanol precipitation.  Deep-sequencing libraries were generated by PCR amplification of sgRNA guide strands using primers that tag the product with standard Illumina adapters and a 4 bp sample barcode in a 2 step-PCR protocol.