Comment[ArrayExpressAccession] E-GEOD-45669 MAGE-TAB Version 1.1 Public Release Date 2013-07-22 Investigation Title RNA-sequencing of the human milk fat layer during colostrum, transitional, and mature stages of lactation Comment[Submitted Name] RNA-sequencing of the human milk fat layer during colostrum, transitional, and mature stages of lactation Experiment Description Human milk fat globules, by enveloping cell contents during their secretion into milk, are a rich source of mammary cell RNA. Here, we pair this non-invasive mRNA source with RNA sequencing technology to probe the milk fat layer transcriptome during three stages of lactation: colostral, transitional, and mature milk production. We find that transcriptional profiles cluster not by postpartum day, but by milk Na:K ratio, indicating that women sampled during the same postpartum time frame could be at markedly different stages of gene expression. Each stage of lactation is characterized by a dynamic range (105-fold) in transcript abundances not previously observed with microarray technology. We discovered that transcripts for isoferritins and cathepsins are strikingly abundant during colostrum production, highlighting the potential importance of these proteins for neonatal health. Two transcripts, encoding M-NM-2-casein (CSN2) and a-lactalbumin (LALBA), make up 45% of the total pool of mRNA in mature lactation. Genes significantly expressed across all stages of lactation are associated with making, modifying, transporting, and packaging milk proteins. Stage-specific transcripts are associated with immune defense during the colostral stage, up-regulation of the machinery needed for milk protein synthesis during the transitional stage, and the production of lipids during mature lactation. We observed strong modulation of key genes involved in lactose synthesis and insulin signaling. In particular, PTPRF may serve as a biomarker linking insulin resistance with insufficient milk supply. This study provides the methodology and reference data set to enable future targeted research on the physiological contributors to sub-optimal lactation in humans. Milk fat mRNA profiles were generated from Day 2 and mature milk samples obtained from lactating mothers Term Source Name ArrayExpress EFO Term Source File http://www.ebi.ac.uk/arrayexpress/ http://www.ebi.ac.uk/efo/efo.owl Person Last Name Lemay Lemay Nommsen-Rivers Person First Name Danielle Danielle Laurie Person Mid Initials G G Person Email dglemay@ucdavis.edu Person Affiliation University of California Davis Person Address Genome Center, University of California Davis, 451 Health Sciences Dr, Davis, CA, USA Person Roles submitter Protocol Name P-GSE45669-2 P-GSE45669-1 Protocol Description Basecalls performed using Illumina software Reads mapped to the genome using TopHat with these options: tophat -G hg19.gtf -o tophat -p 4 -r 0 Mapped reads were indexed using samtools: samtools index tophat/accepted_hits.bam Normalized transcript abundances determined using Cufflinks: cufflinks -u -b hg19.fa -G hg19.gtf -p 4 -o cufflinks tophat/accepted_hits.bam Genome_build: hg19 Supplementary_files_format_and_content: Compressed tab-delimited text files contain transcript abundance measurements (FPKM) summarized at the gene level Soft Spin: Whole milk samples were centrifuged at 300 g for 10 min at 4 degrees C. The milk fat layers from three, 2.0 mL aliquots were transferred into a single new tube to which 500 M-5L TRIzolM-. was added and stored at -80 degrees C Hard Spin: Whole milk samples were centrifuged at 15000 g for 10 min at 4 degrees C. The milk fat layers from three, 2.0 mL aliquots were transferred into a single new tube to which 500 M-5L TRIzolM-. was added and stored at -80 degrees C Wash: After a hard spin at a warmer temperature, (15,000 g at 12 degrees C) the milk fat layers were transferred to a clean set of 3 tubes and re-suspended in 1 mL PBS+10M-5M EDTA. After centrifuging again (15,000 g at 12 degrees C), the milk fat layers from all 3 tubes were transferred to a single tube, re-suspended in 1 mL PBS, and centrifuged a final time (15,000 g at 12 degrees C). The remaining milk fat layer was transferred into a single new tube to which 500 M-5L TRIzolM-. was added and stored at -80 degrees C. RNA Isolation: Total RNA was extracted and purified in batches of 16 using the PROMEGA Maxwell 16M-bM-^DM-" integrated system (Promega Corporation, Madison, WI). RNA libraries were prepared for sequencing using the Illumina TruSeq RNA Kit and standard Illumina Protocols Protocol Type normalization data transformation protocol nucleic acid library construction protocol Experimental Factor Name LACTATION STAGE EXTRACTION PROTOCOL SUBJECT ID Experimental Factor Type lactation stage extraction protocol subject id Publication Title RNA sequencing of the human milk fat layer transcriptome reveals distinct gene expression profiles at three stages of lactation. Publication Author List Lemay DG, Ballard OA, Hughes MA, Morrow AL, Horseman ND, Nommsen-Rivers LA PubMed ID 23861770 Publication DOI 10.1371/journal.pone.0067531 Comment[SecondaryAccession] GSE45669 Comment[GEOReleaseDate] 2013-07-22 Comment[ArrayExpressSubmissionDate] 2013-04-01 Comment[GEOLastUpdateDate] 2013-08-26 Comment[AEExperimentType] RNA-seq of coding RNA Comment[AdditionalFile:Data1] GSE45669_master_allPE_genelevel_FPKM.txt Comment[SecondaryAccession] SRP020470 Comment[SequenceDataURI] http://www.ebi.ac.uk/ena/data/view/SRR801690-SRR801705 SDRF File E-GEOD-45669.sdrf.txt