briefly , we injected 12  ng of morpholino into 4-cell stage x. laevis embryos to knock down rfx2 expression ; the morpholino sequence has been previously reported .
we then prepared 100 animal caps ( ectodermal explants of stage 10 x. laevis embryos , dissected with forceps ) , both for control samples and rfx2 morphants , and cultured them until stage 20 .
the stage of animal caps was estimated by comparison against embryos from the same clutch .
total rna was collected using the trizol method , and then processed using a non - strand - specific illumina rna - seq library preparation kit with poly - a enrichment ( truseq v2 ) .
we sequenced these libraries in a 2    50  bp paired - end configuration using an illumina hiseq 2000 .
the x. laevis genome project was ongoing when we collected these data , so for this study we used a draft genome sequence ( jgi version 6.0 genome scaffolds ; available at ftp://ftp.xenbase.org/pub/genomics/jgi/xenla6.0/ ) and annotation (  oktoberfest version of putative transcripts , mainly derived from rna - seq de novo assembly and then confirmed against jgi version 6.0 genome scaffolds ; see http://www.marcottelab.org/index.php/xenla_oktoberfest for more details ) .
all scaffolds and transcripts are available at xenbase ( ftp://xenbaseturbofrog.org/sequence_information/uta/ ) and our supplementary website ( http://www.marcottelab.org/index.php/chungkwon2013_rfx2 ) . because it is easier for gene - level expression analysis , we conducted rna - seq mapping against putative transcripts rather than the whole genome . using bowtie1 ( version 0.12.7 ) , we mapped our rna - seq reads to the oktoberfest models ( which contain 25,537 putative transcripts for each gene ) using the longest transcript model for each locus .
then we used edger   to identify differentially expressed genes , focusing on genes with greater than 2-fold difference and a false discovery rate less than 0.05 .
one of the challenges in x. laevis rna - seq analysis is the presence of homoeologs , i.e. duplicated genes that arise as a result of allotetraploidy . using an allowance of 2 mismatches within a 50-bp read ( the  -v 2 option in bowtie1 )
we used two datasets for this test : ( 1 ) 827 gene pairs previously identified by a variety of labs and curated at xenbase using an 
-a/-b gene name suffix , and ( 2 ) 2218 assembled est pairs identified as involved in a trio relationship with xenopus tropicalis 
 . as shown in fig .  1 , 6875% of reads
we were particularly interested in the differential expression between wild - type embryos and rfx2 morphants .
thus , in order to maximize the expression signals in our analysis , we allowed for all possible hits in mapping with the  -a option ( i.e. interchangeably mapped reads would be counted twice ) , and then conducted differential expression analysis .
ultimately we found no major differences in differential analysis between these approaches ( data not shown ) .
out of 24,089 x. laevis transcripts detected in our rna - seq experiments , 3209 transcripts were down - regulated in the rfx2 knockdown condition , and 1523 transcripts were up - regulated . to perform functional network analysis using humannet , we converted these gene lists to human orthologs ( based on ensembl version 69 ) .
note that initial orthology assignments are already captured by the x. laevis oktoberfest transcript gene names , because as part of the transcript set construction , all x. laevis protein sequence candidates were compared to the reference proteome of five different species ( human , mouse , zebrafish , chicken , and x. tropicalis ) for the purpose of assigning gene names consistent with the human orthologs . for homoeologs , if only one of two duplicated genes was determined to be significantly differentially expressed but not the other , we still assigned the corresponding human gene as being differentially expressed .
after converting all x. laevis genes into human orthologs , we identified 2750 human candidate genes transcriptionally regulated by rfx2 . the detailed description of our chip - seq sample preparation has been previously reported .
briefly , we injected mrna encoding gfp - tagged rfx2 into 4-cell stage x. laevis embryos and then pulled down the tagged protein with -gfp antibody ( ab290 ) from 600 whole embryos ( stage 20 ) . before immunoprecipitation
, we cross - linked rfx2-genomic dna complexes with 1% formaldehyde and fragmented them with a branson 450 sonifier ( expected fragment size was from 200 to 500  bp ) . as a control , we injected gfp messenger rnas alone and conducted the same immunoprecipitation procedure .
dna fragments were extracted with phenol  chloroform and purified with a qiaquick pcr purification kit ( qiagen ) .
sequencing libraries were prepared with a standard illumina genomic library construction kit ( truseq ) and sequenced with an illumina hiseq 2000 in 1    50  bp configuration .
briefly , we injected 12  ng of morpholino into 4-cell stage x. laevis embryos to knock down rfx2 expression ; the morpholino sequence has been previously reported .
we then prepared 100 animal caps ( ectodermal explants of stage 10 x. laevis embryos , dissected with forceps ) , both for control samples and rfx2 morphants , and cultured them until stage 20 .
the stage of animal caps was estimated by comparison against embryos from the same clutch .
total rna was collected using the trizol method , and then processed using a non - strand - specific illumina rna - seq library preparation kit with poly - a enrichment ( truseq v2 ) .
we sequenced these libraries in a 2    50  bp paired - end configuration using an illumina hiseq 2000 .
the x. laevis genome project was ongoing when we collected these data , so for this study we used a draft genome sequence ( jgi version 6.0 genome scaffolds ; available at ftp://ftp.xenbase.org/pub/genomics/jgi/xenla6.0/ ) and annotation (  oktoberfest version of putative transcripts , mainly derived from rna - seq de novo assembly and then confirmed against jgi version 6.0 genome scaffolds ; see http://www.marcottelab.org/index.php/xenla_oktoberfest for more details ) .
all scaffolds and transcripts are available at xenbase ( ftp://xenbaseturbofrog.org/sequence_information/uta/ ) and our supplementary website ( http://www.marcottelab.org/index.php/chungkwon2013_rfx2 ) . because it is easier for gene - level expression analysis , we conducted rna - seq mapping against putative transcripts rather than the whole genome . using bowtie1 ( version 0.12.7 ) , we mapped our rna - seq reads to the oktoberfest models ( which contain 25,537 putative transcripts for each gene ) using the longest transcript model for each locus .
then we used edger   to identify differentially expressed genes , focusing on genes with greater than 2-fold difference and a false discovery rate less than 0.05 .
one of the challenges in x. laevis rna - seq analysis is the presence of homoeologs , i.e. duplicated genes that arise as a result of allotetraploidy .
using an allowance of 2 mismatches within a 50-bp read ( the  -v 2 option in bowtie1 ) , we evaluated how many reads were mapped interchangeably between homoeologs .
we used two datasets for this test : ( 1 ) 827 gene pairs previously identified by a variety of labs and curated at xenbase using an 
-a/-b gene name suffix , and ( 2 ) 2218 assembled est pairs identified as involved in a trio relationship with xenopus tropicalis 
 . as shown in fig .  1 , 6875% of reads
we were particularly interested in the differential expression between wild - type embryos and rfx2 morphants .
thus , in order to maximize the expression signals in our analysis , we allowed for all possible hits in mapping with the  -a option ( i.e. interchangeably mapped reads would be counted twice ) , and then conducted differential expression analysis .
ultimately we found no major differences in differential analysis between these approaches ( data not shown ) . out of 24,089 x. laevis transcripts detected in our rna - seq experiments ,
3209 transcripts were down - regulated in the rfx2 knockdown condition , and 1523 transcripts were up - regulated . to perform functional network analysis using humannet , we converted these gene lists to human orthologs ( based on ensembl version 69 ) .
note that initial orthology assignments are already captured by the x. laevis oktoberfest transcript gene names , because as part of the transcript set construction , all x. laevis protein sequence candidates were compared to the reference proteome of five different species ( human , mouse , zebrafish , chicken , and x. tropicalis ) for the purpose of assigning gene names consistent with the human orthologs . for homoeologs , if only one of two duplicated genes was determined to be significantly differentially expressed but not the other , we still assigned the corresponding human gene as being differentially expressed .
after converting all x. laevis genes into human orthologs , we identified 2750 human candidate genes transcriptionally regulated by rfx2 .
briefly , we injected mrna encoding gfp - tagged rfx2 into 4-cell stage x. laevis embryos and then pulled down the tagged protein with -gfp antibody ( ab290 ) from 600 whole embryos ( stage 20 ) . before immunoprecipitation , we cross - linked rfx2-genomic dna complexes with 1% formaldehyde and fragmented them with a branson 450 sonifier ( expected fragment size was from 200 to 500  bp ) .
as a control , we injected gfp messenger rnas alone and conducted the same immunoprecipitation procedure .
dna fragments were extracted with phenol  chloroform and purified with a qiaquick pcr purification kit ( qiagen ) .
sequencing libraries were prepared with a standard illumina genomic library construction kit ( truseq ) and sequenced with an illumina hiseq 2000 in 1    50  bp configuration .
similar to the rna - seq data analysis , we conducted chip - seq analysis to discriminate between homoeolog genes .
we applied a more stringent criteria for chip - seq read mapping , requiring uniquely mapped reads to the genome scaffold ( jgi version 6.0 ) and a maximum of 2 mismatches within the seed sequence ( i.e. the  -m 1 -n 2 options in bowtie1 ) . for peak calling , we used macs ( version 1.4.2 ) with default options .
we initially determined significant rfx2-bound peaks by using a false discovery rate ( fdr ) cutoff ( <  0.05 ) reported by macs .
however , as shown in fig .  2 , only a few peaks demonstrated an fdr above 0.05 if the fold enrichment of the peak was greater than 20 , so we included these peaks as well in our further analysis .
for each peak , we assigned the closest protein - coding gene as its target gene , so long as it was within 10  kb .
most of these peaks were located less than 1000  bp from the transcript start site of their assigned gene , suggesting that , if anything , our criteria for associating chip - seq peaks to target genes were over - generous . out of 29,448 peaks identified in total ,
6646 peaks were selected for further study that exhibited either an fdr  <  5% or a fold - enrichment >  20 , and 5024 of those peaks were assigned to their neighboring genes . as with our rna - seq data analysis
, we converted the 5024 x. laevis target gene ids to human genes , collapsing duplicated genes into a single human ortholog based on their names .
this analysis resulted in a final set of 911 putative directly bound rfx2 target genes that also showed significantly differential gene expression after rfx2 knockdown .
a list of all 911 genes is available in supplemental file 1 in our previous report .
 recently , using the frog xenopus laevis as a model system , we showed that the transcription factor rfx2 coordinates many genes involved in ciliogenesis and cell movement in multiciliated cells ( chung et al . , 
 2014 ) . to our knowledge , it was the first paper to utilize the genomic resources , including genome sequences and interim gene annotations , from the ongoing x. laevis genome project . for researchers who are interested in the application of genomics and systems biology approaches in xenopus studies , here 
 we provide additional details about our dataset ( ncbi geo accession number gse50593 ) and describe how we analyzed rna - seq and chip - seq data to identify direct targets of rfx2 . 
Direct link to deposited data
Experimental design, materials and methods
RNA-seq experiments
RNA-seq analysis
ChIP-seq experiments
ChIP-seq analysis