Protocols: Samples were collected at different time points to allow comparisons with the end-point sample, mScarlet+ cells, to track gRNA diversity and enrichment. Puromycin-resistant (PuroR) cells represented the diversity and depth of the sgRNA transduced lentiviral pool. Two samples were collected during hygromycin selection: first, at a starting non-lethal dose (Hy50) to trigger cassette and increase clone representation, then to capture hygromycin-resistant cells (HyR). Finally, mScarlet expressing cells were collected. For sample collection at each mentioned time points, all cells in different dishes were pooled and about 25% of the total cells were harvested, to ensure that we do not create any artificial bottleneck and gRNA diversity is well represented. In addition, lentiviral gRNA plasmid pool used to produce lentivirus represents the starting point. Murine embryonic stem cells J1 (129S4/SvJae) were cultured on gelatin-coated dishes in Serum/LIF medium containing DMEM supplemented with 15% fetal bovine serum (FBS), non-essential amino acids (NEAA), penicillin/streptomycin and 1000 U/ml leukemia inhibitory factor (LIF). Cells were incubated in a humidified atmosphere at 37°C under 5% CO2. The screen was performed in two biological replicates, starting with 140 million cells each; cells were transduced with lentiviral pool at multiplicity of infection (MOI) ~0.1. After 48h, transduced cells were positively selected with 2µg/ml puromycin for 5 days. Following this, cells were initially selected with non-lethal dose of 50µg/ml hygromycin for 3 days followed by additional selection for 11 days at lethal dose of hygromycin (125µg/ml). Cell medium was changed every day with fresh antibiotics. Hygromycin resistant cells were sorted by FACS for mScarlet expressing cells. The gRNA library plasmid DNA was isolated from the bacterial cell pellets using NucleoBond Xtra Maxi kit (MACHEREY-NAGEL) following recommended protocol. Genomic DNA was isolated from cells using AllPrep DNA/RNA Mini Kit (Qiagen) using the recommended protocol for automated QIAcube instrument. The gRNAs were amplified by employing the Broad Institute protocol at Addgene with some modifications. For plasmid library, 50ng DNA was used for each reaction whereas 200ng of genomic DNA was used per reaction. Briefly, polymerase chain reactions (PCR) were performed with Platinum Taq polymerase (ThermoFisher), designed pool of P5 primers and a unique P7 barcode primer (primers sequences are in supplemental data). The PCR conditions were: initial denaturation at 94°C for 4 min; 28 cycles of denaturation at 94°C for 30 s, annealing at 53°C for 30 s and extension at 72°C for 30 s per kb; final extension at 72°C for 10 min. For each sample, four 50µl PCR reactions were performed and pooled afterwards. The PCR product were verified for the right amplicon on agarose gels and DNA was retrieved using QIAquick PCR Purification Kit (Qiagen). The DNA was further purified using AMPure XP (Beckman Coulter)
ENA first public
2022-01-01
ENA last update
2022-01-01
External Id
SAMEA6863003
INSDC center alias
BioSysM - LMU Munich
INSDC center name
BioSysM - LMU Munich
INSDC first public
2022-01-01T00:12:24Z
INSDC last update
2022-01-01T00:12:24Z
INSDC status
public
Submitter Id
E-MTAB-9126:Hy125
broker name
ArrayExpress
cell line
J1
cell type
embryonic stem cell
common name
house mouse
developmental stage
embryo
fraction
unsorted
genotype
Dazl_CGI_Snrpn:HygroR+Dazl:mScarlet
organism part
inner cell mass
sample name
E-MTAB-9126:Hy125
selection method
hygromycin selected