neoplastic cell lines used in this study were te-1 , te-5 , and te-8 , which were established from well- , poorly- , and moderately - differentiated escc , respectively . these cell lines were purchased from riken cell bank ( tsukuba , japan ) and cultured in rpmi-1640 medium supplemented with 10% fetal bovine serum . het-1a , an sv40 large t antigen - harboring normal human esophageal squamous cell line , was purchased from american type culture collection ( manassas , va , usa ) and cultured using the begm kit ( lonza , basel , switzerland ) but without addition of the ga-1000 ( gentamycin - amphotericin b mix ) provided with the kit . total rna was isolated from these cells using isogen reagent ( wako , osaka , japan ) according to the manufacturer 's protocol . rna purity was evaluated by the rna integrity number ( rin ) , a representative index to assess rna quality determined using the agilent 2100 bioanalyzer ( agilent , santa clara , ca , usa ) . the rin value of all rna samples used in this study was shown to be 10 . the rna samples were then subjected to ribosomal and mitochondrial rna depletion using ribo - zero rrna removal kit ( illumina ) . maltiplex rna - seq libraries were generated for samples using truseq stranded total library preparation kit ( illumina ) . sequencing was carried out on hiseq2500 ( illumina ) according to the protocol of 2    125  bp run . as an output of this sequencing , 53 million to 66 million raw reads trimming and filtering of the reads to remove adaptor sequences and low - quality nucleotides were performed using trimmomatic ( ver . 0.32 ) ( http://www.usadellab.org/cms/?page=trimmomatic).table 1overview of sequencing results and statistics.table 1sampleraw readscalled bases ( mbp)%q30aaverage qualityhet-1a53,656,462670793.5535.25te-153,331,576666693.2735.16te-558,934,174736793.2435.16te-865,921,940824093.2835.16aquality score 30 , an index to predict the probability of an error in base calling . overview of sequencing results and statistics . quality score 30 , an index to predict the probability of an error in base calling . for the individual cell lines , 90.392.3% of the filtered reads were mapped to the genome . the statistics of sequencing and mapping results are summarized in table 1 and table 2 , respectively.table 2overview of mapping.table 2sampleraw reads ( m)cleaned reads ( mbp)reads mapped ( m)mapping rates ( % ) het-1a53.744.340.691.5te-153.342.938.890.5te-558.947.042.891.0te-865.954.650.392.1 assembly of transcriptomes from rna - seq data and quantification of their expression levels were performed using cufflinks ( ver . fragments per kilobase of exon per million fragments mapped ( fpkm ) values are used as an index of expression level . 1 ) . using fpkm values of all annotated genes , the values of correlation coefficients between het-1a and te-1/5/8 are calculated to be 0.234 ( het-1a vs. te-1 ) , 0.323 ( het-1a vs. te-5 ) , and 0.401 ( het-1a vs. te-8 ) . correlation coefficients among the three escc - derived cell lines range from 0.419 to 0.697 . these values possibly indicate that the individual escc - derived cell lines show a more similar gene expression pattern to each other than to het-1a . when analysis objects are filtered with the criteria of fpkm >  1 or >  10 for both of paired samples , the values of correlation coefficients increase to 0.8040.956.fig . neoplastic cell lines used in this study were te-1 , te-5 , and te-8 , which were established from well- , poorly- , and moderately - differentiated escc , respectively . these cell lines were purchased from riken cell bank ( tsukuba , japan ) and cultured in rpmi-1640 medium supplemented with 10% fetal bovine serum . het-1a , an sv40 large t antigen - harboring normal human esophageal squamous cell line , was purchased from american type culture collection ( manassas , va , usa ) and cultured using the begm kit ( lonza , basel , switzerland ) but without addition of the ga-1000 ( gentamycin - amphotericin b mix ) provided with the kit . total rna was isolated from these cells using isogen reagent ( wako , osaka , japan ) according to the manufacturer 's protocol . rna purity was evaluated by the rna integrity number ( rin ) , a representative index to assess rna quality determined using the agilent 2100 bioanalyzer ( agilent , santa clara , ca , usa ) . the rin value of all rna samples used in this study was shown to be 10 . the rna samples were then subjected to ribosomal and mitochondrial rna depletion using ribo - zero rrna removal kit ( illumina ) . maltiplex rna - seq libraries were generated for samples using truseq stranded total library preparation kit ( illumina ) . sequencing was carried out on hiseq2500 ( illumina ) according to the protocol of 2    125  bp run . as an output of this sequencing , 53 million to 66 million raw reads trimming and filtering of the reads to remove adaptor sequences and low - quality nucleotides were performed using trimmomatic ( ver . 0.32 ) ( http://www.usadellab.org/cms/?page=trimmomatic).table 1overview of sequencing results and statistics.table 1sampleraw readscalled bases ( mbp)%q30aaverage qualityhet-1a53,656,462670793.5535.25te-153,331,576666693.2735.16te-558,934,174736793.2435.16te-865,921,940824093.2835.16aquality score 30 , an index to predict the probability of an error in base calling . overview of sequencing results and statistics . quality score 30 , an index to predict the probability of an error in base calling . the filtered reads were aligned to the ensembl human genome grch38 . p7 as a reference ( ftp://ftp.ensembl.org/pub/release-85/fasta/homo_sapiens/dna/ ) using bowtie ( ver . the statistics of sequencing and mapping results are summarized in table 1 and table 2 , respectively.table 2overview of mapping.table 2sampleraw reads ( m)cleaned reads ( mbp)reads mapped ( m)mapping rates ( % ) het-1a53.744.340.691.5te-153.342.938.890.5te-558.947.042.891.0te-865.954.650.392.1 assembly of transcriptomes from rna - seq data and quantification of their expression levels were performed using cufflinks ( ver . 2.2.1 ) . fragments per kilobase of exon per million fragments mapped ( fpkm ) values are used as an index of expression level . 1 ) . using fpkm values of all annotated genes , the values of correlation coefficients between het-1a and te-1/5/8 are calculated to be 0.234 ( het-1a vs. te-1 ) , 0.323 ( het-1a vs. te-5 ) , and 0.401 ( het-1a vs. te-8 ) . correlation coefficients among the three escc - derived cell lines range from 0.419 to 0.697 . these values possibly indicate that the individual escc - derived cell lines show a more similar gene expression pattern to each other than to het-1a . when analysis objects are filtered with the criteria of fpkm >  1 or >  10 for both of paired samples , the values of correlation coefficients increase to 0.8040.956.fig .