Illumina HiSeq 2500 paired end sequencing; CRISPR-Cas9 screening of lncRNAs and protein coding genes involved in the transdifferentiation from human pre-B cells into macrophages
CRISPR-Cas9 screening of lncRNAs and protein coding genes involved in the transdifferentiation from human pre-B cells into macrophages
H072_delayed_R2_p
Cells were collected at different time points after transdifferentiation induction: 0, 72 and 144 hours. Cells were sorted by Fluorescence Analysis Cell Sorting (FACS) prior to DNA extraction. Human BLaER1 cells were grown in RPMI medium (Invitrogen) supplemented with 10% heat-inactivated foetal bovine serum (FBS, Gibco), 2 mM L-glutamine, and 100 U/ml penicillin G sodium (Gibco). Cells were maintained at 37ºC in a humid atmosphere containing 5% CO2. Transdifferentiation was induced by addition of 100 nM of beta-estradiol (Calbiochem) and 10 nM of IL-3 and CSF-1 (Peprotech). Genomic DNA was extracted from the FACS sorted cells with the GeneJET Genomic DNA purification kit (Thermo Scientific) following the instructions of the manufacturer. A first PCR step was done by Phusion polymerase (Thermo Fisher) using 500 ng of genomic DNA and staggered oligo mix with the presence of 6% DMSO, annealing temperature of 60ºC and a total of 20 cycles of amplification. We used staggered oligos to avoid the same bases being read for the constant region during Illumina sequencing and to minimize technical issues during base calling. Up to 6 PCR reactions were combined, the amplicons were gel-purified, and 2 ng were used as a template for a second PCR. The second PCR step was also done by Phusion polymerase but without the presence of DMSO. We used Illumina barcoded oligos an annealing temperature of 60ºC and a total of 8 cycles of amplification. Samples were purified with Agencourt Ampure beads (Beckman Coulter), quantified with a Qubit fluorometer (Thermo Scientific) and checked for quality in a Bioanalyzer (Agilent).