kh2 mouse embryonic stem cell ( esc ) line is used as wild type cells . next , an exogenous erk1 gene is integrated into the engineered cola1 locus through flpe recombinase - mediated recombination , resulting in a doxycycline ( dox)-inducible erk1 transgene ( ierk1 ; erk1 escs ) . in the presence of dox , both endogenous erk2 alleles in ierk1 ; erk1 cells are disrupted by cas9 . the resulting cell line is named ierk1 ; erk ko escs . when cultured in medium supplemented with dox thus , ierk1 ; erk ko escs cultured in the absence of dox for longer than 48  h are considered as erk ko escs . to gain insights into the role of erk signaling in mek inhibition of escs , six samples , wt kh2 escs treated with or without pd for 48  h ( kh2_pd and kh2 , respectively ) , ierk1 ; erk ko escs cultured with dox ( p0 ) , 48 and 96  h after dox withdrawal ( p1 and p2 , respectively ) , and ierk1 ; erk ko escs cultured without dox for 96  h and treated with pd in the last 48  h ( p2_pd ) , are subjected to rna sequencing ( rna - seq ) analysis . total rna is extracted from cells using rneasy mini kit ( qiagen ) . on - column dnase i digestion ( dnase - free dnase set , qiagen ) is performed according to manufacturer protocols to eliminate genomic dna contamination . then the mrna is enriched with the oligo(dt ) magnetic beads ( for eukaryotes ) , and is fragmented into short fragments ( about 200  bp ) . with random hexamer - primer , the first strand of cdna is synthesized , and then the second strand is synthesized . the ends of the double strand cdna are repaired , and a single nucleotide a ( adenine ) is added to the 3-ends . finally , sequencing adaptors are ligated to the fragments . the ligation products are amplified with pcr . for quality control , rna and library preparation integrity are verified using agilent 2100 bioanalyzer system and abi steponeplus real - time pcr system . standard barcoded rna - seq libraries are generated for sequencing with illumina hiseqtm 2000 ( se50 ) . construction of rna sequencing library , sequencing with illumina hiseqtm 2000 , and bioinformatic analysis are performed by bgi tech ( bgi , shenzhen , china ) . by base calling , the original image data produced by the sequencer is translated into sequences , which are defined as raw data is filtered to remove sequences of low quality , and only high quality reads are retained as the clean reads ( clean data ) and used for the subsequent analysis . the filtering procedure includes the following three steps : ( a ) remove reads with adaptor sequences . ( b ) remove reads in which the percentage of unknown bases ( n ) is greater than 10% . ( c ) remove low quality reads . if the percentage of the low quality base ( base with quality value   5 ) is greater than 50% in a read , we define this read as low quality . reads per kilobase per million reads ( rpkm ) is calculated to represent the gene expression level , and saved as  .gene.rpkm.xls  files . the formula is rpkm  =  10    c/(n    l/10 ) . here rpkm(a ) is the expression level of gene a , c is the number of reads that uniquely aligned to gene a , n is the total number of reads that uniquely aligned to all genes , and l is the number of bases of gene a. if there is more than one transcript for a gene , the longest one is used to calculate its expression level and coverage . the criteria of more than two fold change and false discovery rate ( fdr ) <  0.001 are used to identify lists of differentially expressed genes ( degs ) in each compared group . three lists of degs , meki ( wt ) , meki ( erk ko ) , and erk ko , are generated by paired comparisons of kh2_pd and kh2 , p2_pd and p2 , and p1 and p0 , respectively ( fig . . further comparison of these three lists of degs reveals that erk ko are quite different from meki ( wt ) ( fig . 1b ) , consistent with our observation that erk ko and mek inhibition have distinct effect on the self - renewal and proliferation of mouse escs . moreover , meki ( erk ko ) and meki ( wt ) share a significant portion of genes ( fig . 1c ) , suggesting that these genes are regulated by mek inhibition , regardless of the presence or absence of erk . kh2 mouse embryonic stem cell ( esc ) line is used as wild type cells . next , an exogenous erk1 gene is integrated into the engineered cola1 locus through flpe recombinase - mediated recombination , resulting in a doxycycline ( dox)-inducible erk1 transgene ( ierk1 ; erk1 escs ) . in the presence of dox , both endogenous erk2 alleles in ierk1 ; erk1 cells are disrupted by cas9 . the resulting cell line is named ierk1 ; erk ko escs . when cultured in medium supplemented with dox thus , ierk1 ; erk ko escs cultured in the absence of dox for longer than 48  h are considered as erk ko escs . to gain insights into the role of erk signaling in mek inhibition of escs , six samples , wt kh2 escs treated with or without pd for 48  h ( kh2_pd and kh2 , respectively ) , ierk1 ; erk ko escs cultured with dox ( p0 ) , 48 and 96  h after dox withdrawal ( p1 and p2 , respectively ) , and ierk1 ; erk ko escs cultured without dox for 96  h and treated with pd in the last 48  h ( p2_pd ) , are subjected to rna sequencing ( rna - seq ) analysis . total rna is extracted from cells using rneasy mini kit ( qiagen ) . on - column dnase i digestion ( dnase - free dnase set , qiagen ) is performed according to manufacturer protocols to eliminate genomic dna contamination . then the mrna is enriched with the oligo(dt ) magnetic beads ( for eukaryotes ) , and is fragmented into short fragments ( about 200  bp ) . with random hexamer - primer , the first strand of cdna is synthesized , and then the second strand is synthesized . the double strand cdna is purified with magnetic beads . the ends of the double strand cdna are repaired , and a single nucleotide a ( adenine ) is added to the 3-ends . finally , sequencing adaptors are ligated to the fragments . the ligation products are amplified with pcr . for quality control , rna and library preparation integrity are verified using agilent 2100 bioanalyzer system and abi steponeplus real - time pcr system . standard barcoded rna - seq libraries are generated for sequencing with illumina hiseqtm 2000 ( se50 ) . construction of rna sequencing library , sequencing with illumina hiseqtm 2000 , and bioinformatic analysis are performed by bgi tech ( bgi , shenzhen , china ) . by base calling , the original image data produced by the sequencer is translated into sequences , which are defined as  raw reads(or  raw data  ) and saved as  .fastq raw data is filtered to remove sequences of low quality , and only high quality reads are retained as the clean reads ( clean data ) and used for the subsequent analysis . the filtering procedure includes the following three steps : ( a ) remove reads with adaptor sequences . ( b ) remove reads in which the percentage of unknown bases ( n ) is greater than 10% . ( c ) remove low quality reads . if the percentage of the low quality base ( base with quality value   5 ) is greater than 50% in a read , we define this read as low quality . reads per kilobase per million reads ( rpkm ) is calculated to represent the gene expression level , and saved as  .gene.rpkm.xls  files . the formula is rpkm  =  10    c/(n    l/10 ) . here rpkm(a ) is the expression level of gene a , c is the number of reads that uniquely aligned to gene a , n is the total number of reads that uniquely aligned to all genes , and l is the number of bases of gene a. if there is more than one transcript for a gene , the longest one is used to calculate its expression level and coverage . the criteria of more than two fold change and false discovery rate ( fdr ) <  0.001 are used to identify lists of differentially expressed genes ( degs ) in each compared group . three lists of degs , meki ( wt ) , meki ( erk ko ) , and erk ko , are generated by paired comparisons of kh2_pd and kh2 , p2_pd and p2 , and p1 and p0 , respectively ( fig . . further comparison of these three lists of degs reveals that erk ko are quite different from meki ( wt ) ( fig . 1b ) , consistent with our observation that erk ko and mek inhibition have distinct effect on the self - renewal and proliferation of mouse escs . moreover , meki ( erk ko ) and meki ( wt ) share a significant portion of genes ( fig . 1c ) , suggesting that these genes are regulated by mek inhibition , regardless of the presence or absence of erk .
mek inhibition and erk knockout ( ko ) have quite distinct effects on pluripotency maintenance in mouse embryonic stem cells ( escs ) . to test whether there is an erk - independent function of mek , rna - sequencing ( rna - seq ) 
 is carried out on six samples , wt kh2 escs treated with or without pd0325901 ( pd ) for 48  h ( kh2_pd and kh2 , respectively ) , ierk1 ; erk ko escs cultured in the presence of dox ( p0 ) , 48 and 96  h after dox withdrawal ( p1 and p2 , respectively ) , and ierk1 ; erk ko escs cultured without dox for 96  h , and treated with pd in the last 48  h ( p2_pd ) . 
 these rna - seq data demonstrate that mek inhibition has quite different effect on the transcriptional profile of mouse escs , compared to erk ko . 
 moreover , a significant fraction of genes is regulated by mek inhibition , regardless of the presence or absence of erk , indicating an erk - independent function of mek . 
 rna - seq data are deposited in gene expression omnibus ( geo ) datasets under accession number gse70304 .