Illumina HiSeq 4000 paired end sequencing; RNA-seq expression profiling of OCI-AML2 and OCI-AML3 cells upon CRIPSR/Cas9 –mediated disruption of DDX3X RNA binding domain
RNA-seq expression profiling of OCI-AML2 and OCI-AML3 cells upon CRIPSR/Cas9 –mediated disruption of DDX3X RNA binding domain
OCI-AML2/Cas9 and OCI-AML3/Cas9 cells (stably expressing Cas9) were spin-infected with lentiviral particles expressing pKLV2- U6gRNA5 (BbsI)-PKGpuro2ABFP-W plasmid. This plasmid expresses both puromycin resistance cassette and a specific sgRNA. sgRNAs were designed to target sequences of the DDX3X gene encoding for the two portions of the bipartite DDX3X RNA binding domain (named RNA binding domain A- “RBDA” and RNA binding domain B - “RBDB”). The plasmid expressing no sgRNA was use to produce control cell lines (“empty vector” condition).  Infected cells were cultured in puromycin since day 4 after infection and harvested at day 8 after infection (day 4 after puromycin selection). All conditions were produced in three independent biological replicates.  Total RNA was purified from cells using miRNeasy Mini Kit (Qiagen) following manufacturer instructions. Excess of contaminating DNA was removed by On-Column DNase Digestion with the RNase-Free DNase Set (Qiagen), following manufacturer's instructions. Ribosomal RNA was depleted with Ribo-Zero rRNA Removal Kit (Human/Mouse/Rat) from Illumina(R), following manufacturer's instructions. All conditions were produced in three independent biological replicates.  RNA-seq libraries were produced with NEXTflex RNA-Seq Kit for Illumina Sequencing from Bioo Scientific, following manufacturer's instructions.  The following Illumina barcodes were used.  Sample Sequence TruSeq_Code  OCIAML_2_Empty_REP1 CGATGT A002 OCIAML2_RBDA_REP1 TGACCA A004 OCIAML2_RBDB_REP1 ACAGTG A005 OCIAML_3_Empty_REP1 GCCAAT A006 OCIAML_3_RBDA_REP1 CAGATC A007 OCIAML_3_RBDB_REP1 CTTGTA A012 OCIAML_2_Empty_REP_2 ATCACG A001 OCIAML2_RBDA_REP2_ TTAGGC A003 OCIAML2_RBDB_REP2 ACTTGA A008 OCIAML_3_Empty_REP2 GATCAG A009 OCIAML_3_RBDA_REP2 TAGCTT A010 OCIAML_3_RBDB_REP2 GGCTAC A011 OCIAML_2_Empty_REP3 AGTCAA A013 OCIAML2_RBDA_REP3 AGTTCC A014 OCIAML2_RBDB_REP3 ATGTCA A015 OCIAML_3_Empty_REP3 CCGTCC A016 OCIAML_3_RBDA_REP3 GTGAAA A019 OCIAML_3_RBDB_REP3 GTCCGC A018   RNA-seq libraries were produced with NEXTflex RNA-Seq Kit for Illumina Sequencing from Bioo Scientific, following manufacturer's instructions.
Experimental Factor: cell line
OCI-AML3
Experimental Factor: phenotype
CRISPR/Cas9 mediated knockout of RBDB