title: Control_1

description: CTR ND_1

hyb_protocol: A total of 825 ng Cy3-labeled cRNA (specific activity >6 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 55 microL containing 0.5x Agilent fragmentation buffer and 2x Agilent gene expression blocking agent following the manufacturers instructions.  On completion of the fragmentation reaction, 55 microL of 2× Hi-RPM Hybridization Buffer was added to the fragmentation mixture and hybridized to Agilent C. elegans (V2) 4×44K Gene Expression Microarrays (G2519F-020186) for 17 hours at 65°C in a rotating Agilent hybridization oven.  After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent), and dried by slowly removing from Wash buffer 2. A total of 1.65 micrograms Cy3-labelled cRNA

data_processing: Raw microarray hybridization intensity data from four separate experiments were log-transformed. Three separate probe preparations were used individually or combined in equal amounts for the fourth hybridization. Raw microarray data were log transformed to yield z-scores. The z-ratio was calculated as the difference between the observed gene z-scores for the experimental and the control comparisons, and dividing by the standard deviation associated with the distribution of these differences. Z-ratio values of ± 1.5 were used as cut-off values and calculated using a 5% false discovery rate (FDR) threshold. The complete set was tested for gene set enrichment using parametric analysis of gene set enrichment. For each pathway z-score, a p-value was computed using JMP 6.0 software to test for the statistical significance.

source_name: WT worm_untreated

growth_protocol: Caenorhabditis elegans (C. elegans) nematodes were cultured at 25°C on solid agar plates of standard nematode growth medium (NGM) with OP50 food source unless otherwise, and maintained following standard protocols previously described. The Bristol strain (wild type) and CB5600 ccIs4251[pSAK2 (myo-3p::gfp-lacz(NLS)) + psak4 (myo-3p::mitochondrial gfp)I were from Caenorhabditis Genetics Center (CGC). The mitophagy reporter strain N2;Ex(pmyo-3::dsred::lgg-1;pdct-1::dct-1::gfp) was from Prof. N. Tavernarakis (University of Crete, Greece).

extract_protocol: Four independent experiments per condition were collected, and stored at -80oC in lysis reagent (Qiagen Cat. no. 75142). Crude extracts were prepared by bead beating packed worm pellets with the Mini-Beadbeater 8 (Biospec Products, Oklahoma), and total RNA isolated using RNeasy Mini Kit on the QIAcube according to the manufacturer's protocol (Qiagen, California). RNA concentration and quality was measured by Nanodrop (ThermoFisher, Waltham, MA USA) and the Agilent Bioanalyzer RNA 6000 Chip (Agilent, Santa Clara, CA).

treatment_protocol: N2 worms from the L4 stage were exposed to either vehicle (DMSO) or 25 μM tomatidine, and were collected on adult Day 7 by washing off the plates with M9 buffer.


title: Age 33-35 at donation + low ovarian reserve Sample 1 [D6.1]

status: Public on Aug 29 2017

type: RNA

city: Barcelona

first: Montse

last: BARRAGAN

state: Select One

address: Baldiri Reixac, 15

country: Spain

institute: Clinica EUGIN

description: CELs transformed using GCCN-SST with gain_lock=0.75, then processed using RMA-Sketch. These analysis capabilities are available in Affymetrix Power Tools 1.18.0

hyb_protocol: Affymetrix's standard protocol

scan_protocol: AGCC Standard Protocol / GCS 3000

data_processing: Samples were processed using Affymetrix Power Tools (APT) utilizing GCCN-SST RMA with gain lock percent set to 0.75

label: biotin

molecule: total RNA

organism: Homo sapiens

source_name: Mature MII oocytes

label_protocol: 8 µg cDNA (3.2 µg from the first round and 4.8 µg from the second) were subsequently fragmented by DNAseI and biotinylated by terminal transferase (GeneChip Mapping 250K Nsp assy kit; Affymetrix, CA, USA).

extract_protocol: The zona pellucida was removed by Pronase (Roche Diagnostics, Spain) and oocytes were immediately and individually placed in 45 µl of a Proteinase K-based lysis buffer (20 mM DTT, 0.5% SDS, 1 μg/μl proteinase K, 10 mM Tris.HCl, pH 7.4), incubated at 65ºC for 15 minutes, and stored at -80ºC until use (González-Roca, 2010).   Total RNA extraction and amplification were based on the Pico-Profiling protocol and successive improvements for single oocyte use (Gonzalez-Roca et al, 2010; Vassena et al, 2011). Pico-profiling faithfully amplifies the total RNA from as little as 10 somatic cells, or 1 MII oocyte, with a level of false positive signal in downstream analysis that is equivalent to that obtained without an amplification step. Briefly, RNA was purified individual oocytes using RNA Clean XP bead suspension (Agencourt Bioscience, La Jolla, CA, USA) and eluted in 20 µl water. The RNA preparation was used for library preparation and amplification by Whole Transcriptome Amplification (WTA2, Sigma-Aldrich, Spain). SYBRGreen (Sigma-Aldrich, Spain) was added to the amplification reaction, which was performed in a CFX Real-time instrument (Bio-Rad, CA, USA) to monitor amplification yield. The first amplification reaction was stopped when the SYBRGreen signal reached a plateau, and cDNA was purified and quantified on a Nanodrop ND-1000 spectrophotometer (Thermo-Fischer, MA, USA). A second round of amplification was performed from 10 ng of cDNA.

treatment_protocol: Mature MII oocytes were vitrified on the day of retrieval using an open method and were warmed for the study according to standard procedure (Cryotop®, Kitazato®, BioPharma Co., Ltd; Japan) in five randomized groups, on different days during a period of 14 days, to minimize bias due to the handling process. After warming, oocytes were left for 2 hours at 37ºC and 6% CO2, to allow for reassembly of the metaphase plate (Bromfield et al, 2009).

tag: age

value: Age 33-35 at donation

tag: ovarian reserve

value: low ovarian reserve


title: Tomatidine_1

status: Public on Aug 21 2017

city: Baltimore

first: Supriyo

last: De

state: Maryland

address: 251 Bayview Blvd

department: Laboratory of Genetics and Genomics

country: USA

institute: NIA-IRP, NIH

description: TOM 300

scan_protocol: Following posthybridization rinses, arrays were scanned using an Agilent SureScan microarray Scanner at 5 micron, and hybridization intensity data extracted from the scanned images using Agilent’s Feature Extraction Software 9.1 (Agilent) using default parameters to obtain background subtracted and spatially detrended Processed Signal intensities.

organism: Caenorhabditis elegans

source_name: WT worm_tomatidine

label_protocol: Cyanine-3 (Cy3) labeled cRNA was prepared from 200 ng total RNA using the Low-Input Quick Amp Labeling Kit (Agilent, Santa Clara, CA) according to the manufacturer's instructions, followed by RNeasy column purification (QIAGEN, Valencia, CA).  Dye incorporation and cRNA yield were evaluated with the NanoDrop ND-1000 Spectrophotometer (Wilmington, DE).

tag: treatment

value: Tomatidine_25 micromolar


title: Control_4

description: CTR fem


title: Tomatidine_2

description: TOM 450


title: Control_2

description: CTR 300


title: Tomatidine_3

description: TOM fem


title: Skeletal muscle_Young_replicate_2_human

status: Public on Sep 20 2017

description: Sample name: Y-308

hyb_protocol: Standard Illumina protocol. In short, a total of 0.75 μg of biotin-labeled cRNA was hybridized at 58°C for 16 hours to Illumina's HumanHT-12 V4.0 Expression Bead Chips (Illumina, San Diego, CA). Each BeadChip has more than 47,000 probes derived from the National Center for Biotechnology Information Reference Sequence (NCBI) RefSeq Release 38 (November 7, 2009) and other sources. The arrays were washed, blocked and the biotin labeled cRNA was detected by staining with streptavidin-Cy3.

scan_protocol: Arrays were scanned at a resolution of 0.8 μm using the Illumina BeadArray scanner.

data_processing: Data was extracted using the Illumina GenomeStudio software V2011.1, Gene expression module 1.9.0. Any spots at or below the background were filtered out using an Illumina detection Pvalue of 0.02 and above. The natural log of all remaining scores were used to find the avg and std of each array and the z-score normalization was calculated and presented below.  Z-score = (raw value - avg)/std.  Complete data including detection scores is supplied. ID_REF = Unique identifiers from GPL10558; Z_VALUE = Z transformation of the natural log of the raw intensity values; Detection_Pval = Detection Pvalue from Illumina GenomeStudio software V2011.1, Gene expression module 1.9.0.

source_name: Skeletal muscle_Young

label_protocol: Standard Illumina protocol using Illumina TotalPrep RNA Amplification Kit (Ambion; Austin, TX, cat # IL1791) In short, 0.5 μg of total RNA was first converted into single-stranded cDNA with reverse transcriptase using an oligo-dT primer containing the T7 RNA polymerase promoter site and then copied to produce double-stranded cDNA molecules. The double stranded cDNA was cleaned and concentrated with the supplied columns and used in an overnight in-vitro transcription reaction where single-stranded RNA (cRNA) was generated and labeled by incorporation of biotin-16-UTP.

extract_protocol:
Experimental protocol: Young (31.2±0.86 y), middle-aged (46.0±0.71 y), and old (74.0±4.38 y) male skeletal muscle samples were obtained from humans who underwent surgery for hip dysplasia. Written informed consent was obtained from those participants, and the study was approved by the medical ethics committee of the Rizzoli Hospital (Italy). Muscle samples were flash frozen in liquid nitrogen and stored at -80oC until assayed.
RNA was isolated from skeletal muscle using Trizol Reagent (Invitrogen, Carlsbad, CA) following the manufacturer’s instructions.

tag: tissue

value: Muscle

value: Young

tag: gender

value: male


title: Control_3

description: CTR 450


title: Thymus_WT_replicate_1

scan_protocol: Arrays were scanned at a resolution of 0.8 μm using the Illumina BeadStation 500X scanner.

data_processing: Data was extracted using the Illumina GenomeStudio software V2011.1, Gene expression module 1.9.0. Any spots at or below the background were filtered out using an Illumina detection Pvalue of 0.02 and above. The natural log of all remaining scores were used to find the avg and std of each array and the z-score normalization was calculated and presented below.  Z-score = (raw value - avg)/std.  Complete data including detection scores is supplied.

organism: Mus musculus

label_protocol: Standard Illumina protocol using Illumina TotalPrep RNA Amplification Kit (Ambion; Austin, TX, cat # IL1791) In short, IP RNA was first converted into single-stranded cDNA with reverse transcriptase using an oligo-dT primer containing the T7 RNA polymerase promoter site and then copied to produce double-stranded cDNA molecules. The double stranded cDNA was cleaned and concentrated with the supplied columns and used in an overnight in-vitro transcription reaction where single-stranded RNA (cRNA) was generated and labeled by incorporation of biotin-16-UTP.

extract_protocol:
All animal studies performed were approved according to the guidelines of the NIA Animal Care and Use Committee. Mice were maintained in a 12h light/dark cycle on an ad libitum regular diet. Male GIT2-/- (GIT2KO) (n=3-16) and C57BL/6 WT mice (n=3-8), at 1, 3 and 12 months of age, were bred in the NIA animal facility. Lifespan measurements at this facility for the wild-type (WT) C57/BL6 indicated that for both male and female GIT2KO mice presented a significantly-reduced total lifespan (n≥20 per group:  male WT: 609.3±24.2 days; female WT: 617.25±30.15; male GIT2-/-: 503.15±32; female GIT2-/-: 529.35±21.4). They were fasted 12h before experimentation, sacrificed by carbon dioxide asphyxiation and whole body and organ weights were recorded. Equal numbers of animals belonging to the control and/or experimental groups were experimented upon on the same day. Overall, the order of animal experimentation was performed in a random manner.
Total RNA was isolated from 12 month old mouse spleen, thymus, and lymph nodes (extra, inguinal and mesenteric) with Trizol® (Invitrogen) according to the manufacturers instructions.

value: thymus

tag: mouse strain

tag: genotype

value: WT

value: Male


title: Thymus_WT_replicate_1_technical_replicate


title: Thymus_WT_replicate_3


title: Thymus_WT_replicate_2


title: atm1_Nicotinamide riboside_Day1_Replicate 2

status: Public on Dec 20 2017

description:
ATM1_NR_DAY1_2
Included in analysis.

hyb_protocol: A total of 1.65 micrograms Cy3-labeled cRNA (specific activity >6 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 55 microL containing 0.5x Agilent fragmentation buffer and 2x Agilent gene expression blocking agent following the manufacturer's instructions. On completion of the fragmentation reaction, 55 microL of 2x Hi-RPM Hybridization Buffer was added to the fragmentation mixture and hybridized to Agilent-020186 C. elegans (V2) 4x44K Gene Expression Microarrays (G2519F-020186) for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent), and dried by slowly removing from Wash buffer 2.

scan_protocol: Slides were scanned immediately after washing on the Agilent SureScan Microarray Scanner using one color scan setting for 4x44k array slides (Scan resolution 5 microns, Dye channel: Green and Green PMT: 100%).

data_processing: The scanned images were analyzed with Feature Extraction Software 9.1 (Agilent) using default parameters to obtain background subtracted and spatially detrended Processed Signal intensities.

source_name: atm1 worm, NR-treated, day1

label_protocol: Cyanine-3 (Cy3) labeled cRNA was prepared from 300 ng total RNA using the Low Input Quick Amp Labeling Kit (Agilent, Santa Clara, CA) according to the manufacturer's instructions, followed by RNeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were evaluated with the NanoDrop ND-1000 Spectrophotometer (Wilmington, DE).

growth_protocol: C. elegans worms from a wild-type strain (N2) and atm1 mutated (atm1) were cultured in the presence of 5-Fluoro-deoxyuridine (5-FdUrd) to prevent development of progeny and produce a synchronized population.

extract_protocol: Worms were washed off the plates with M9 buffer from 4 independent experiments and stored at -80 oC in lysis reagent (Qiagen Cat. no. 75142). Crude extracts were prepared by bead beating packed worm pellets with a Mini-Beadbeater 8 (Biospec Products). Total RNA was isolated using RNeasy Mini Kit (Qiagen, Cat.74106) on the QIAcube according to the manufacturer's protocol. RNA integrity and quantity were evaluated using the Agilent Bioanalyzer RNA 6000 chip (Agilent).

treatment_protocol: L4-stage N2 (wild-type) and atm-1 mutated (atm1) C. elegans worms were treated with NAD+ precursor nicotinamide riboside (NR), SIRT1 inhibitor SRT1720 or PARP1 inhibitor Olaparib for 1 or 10 days.

tag: strain/background

tag: genotype/variation

value: nicotinamide riboside

tag: concentration

value: 500 microM

tag: days after treatment


title: Spleen_WT_replicate_3_technical_replicate

value: spleen


title: Thymus_GIT2KO HO_replicate_3

source_name: Thymus_GIT2KO HO

value: GIT2KO homozygote


title: Spleen_WT_replicate_2


title: Spleen_GIT2KO_HO_replicate_1


title: Spleen_WT_replicate_3


title: Spleen_GIT2KO_HO_replicate_3


title: Spleen_WT_replicate_1


title: Spleen_GIT2KO_HO_replicate_2


title: Thymus_GIT2KO HO_replicate_1


title: Mesenteric_lymph_nodes_WT_replicate_3

value: mesenteric lymph nodes


title: Thymus_GIT2KO HO_replicate_2


title: Mesenteric_lymph_nodes_GIT2KO_HO_replicate_3


title: Ingual_lymph_nodes_WT_replicate_1

value: inguinal lymph nodes


title: Mesenteric_lymph_nodes_GIT2KO_HO_replicate_2


title: Ingual_lymph_nodes_WT_replicate_3


title: Mesenteric_lymph_nodes_WT_replicate_2


title: Ingual_lymph_nodes_GIT2KO_HO_replicate_3


title: Mesenteric_lymph_nodes_GIT2KO_HO_replicate_1_technical replicate


title: Mesenteric_lymph_nodes_GIT2KO_HO_replicate_1


title: Ingual_lymph_nodes_GIT2KO_HO_replicate_2


title: Mesenteric_lymph_nodes_WT_replicate_1


title: Ingual_lymph_nodes_WT_replicate_2


title: Ingual_lymph_nodes_GIT2KO_HO_replicate_1


title: Ingual_lymph_nodes_GIT2KO_HO_replicate_2_tecnical replicate


title: BIP2

type: SRA

first: Suvarna

last: Nadendla

state: MD

address: 670 W.Baltimore Street

department: Institute for Genome Sciences

institute: University of Maryland School of Medicine

library_source: transcriptomic

data_processing:
Illumina reads were mapped to the Streptococcus pneumoniae TIGR4 genome using Bowtie v. 0.12.7
The mapped reads were counted using HTSeq and normalized and then the differential expression of each gene for one sample compared to another was determined using DESeq v. 1.5.24, and statistical analyses were performed using R-2.15.2, and was represented as the log2 of the fold-change values.
RPKMs for each gene were calculated for each sample.
Genome_build: NC_003028.3
Supplementary_files_format_and_content: Tab-delimited text files include RPKMs

Illumina reads were mapped to the Mus musculus GRCm38 genome using TopHat v. 1.4.0
The mapped reads were counted using HTSeq and normalized and then the differential expression of each gene for one sample compared to another was determined using DESeq v. 1.5.24, and statistical analyses were performed using R-2.15.2, and was represented as the log2 of the fold-change values.
RPKMs for each gene were calculated for each sample.
Genome_build: Mus musculus GRCm38
Supplementary_files_format_and_content: Tab-delimited text files include RPKMs

organism: Streptococcus pneumoniae TIGR4

source_name: Streptococcus pneumoniae cells, infected mouse blood sample

growth_protocol: Pneumococci were grown in Todd Hewitt Broth (THB) (Acumedia, Neogen) with 0.5% yeast extract (THY) at 37Â°C in 5% CO2 until reaching mid-exponential phase [Optical density, O.D.620nm = 0.5; ~1.0 x 108 colony forming units (CFU)/mL] and serially diluted in sterile phosphate buffered saline (PBS). Blood was collected from anesthetized mice retro-orbitally and transferred to heparin-coated collection tubes. Following euthanasia, hearts were surgically excised and washed in PBS to remove blood. Isolated hearts were homogenized in 5ml of PBS followed by filtration of the homogenate through a 40µm cell strainer. Paired blood (BIP) and strained heart (HIP) samples were flash-frozen at -80° C in working aliquots with 10% glycerol. excised and washed in PBS to remove blood. Isolated hearts were homogenized in 5ml of PBS followed by filtration of the homogenate through a 40Âµm cell strainer. Paired blood (BIP) and strained heart (HIP) samples were flash-frozen at -80Â° C in working aliquots with 10% glycerol.

extract_protocol:
For HIP-RNA, hearts were excised from HIP-infected mice (n=3) when deemed moribund. Hearts were rinsed, diced, fragments washed with ice cold PBS, and the fragments homogenized in RNAprotect bacteria reagent (Qiagen). For BIP-RNA, 10 mice pre-depleted for neutrophils using anti-Ly6G antibody (BioXCell, clone RB6-8C5), were infected with TIGR4 and blood was collected in RNAprotect bacteria reagent (Qiagen) when the mice were deemed moribund such that blood from 5 mice were pooled as one sample (n=2 BIP samples). These heart homogenates were stored at -80°C. On the day of total RNA isolation from heart homogenates, the samples were thawed and spun down to discard the supernatant. The pellets were further homogenized in 600 μL RLT with B-ME buffer using a motorized mortar for 30 seconds. The re-homogenized samples were then disaggregated in a Qiashredder followed by RNA extraction with the RNeasy Micro Kit (Qiagen) with DNase treatment on column and in solution. The isolated RNA was quantitated using Nanodrop and Bioanalyzer. Samples were then depleted of rRNAs using the Ribo-Zero rRNA Removal Kit for Gram-positive bacteria and human/mouse/rat (Illumina, San Diego, CA). For the in vitro biofilm and planktonic samples: planktonic mid-log phase (OD620nm = 0.5) TIGR4 grown in THB were used to seed continuous once-flow through biofilm reactors. Biofilms were allowed to grow for 48 hours prior to collection of bacteria. RNA was isolated from the planktonic seed cultures and their respective biofilms. In total, 3 independent biological samples were collected with planktonic and biofilm RNAs from each experiment paired. Total RNA was extracted from each replicate separately using enzymatic lysis of pneumococcal cells (10 μL mutanolysin, 20 μL proteinase K, 15 μL lysozyme, and 55 μL TE) followed by RNA extraction using the same protocol mentioned above. Blood samples were isolated in a similar manner as biofilm and planktonic samples, including enzymatic lysis, except that cells were first disaggregated with the Qiashredder like for the heart samples.
RNA libraries were prepared for sequencing using standard Illumina protocols

tag: sample type

value: infected mouse blood


title: BIP1


title: Vastus lateralis muscle_Middle-aged_replicate_3_rhesus

description: Sample name: MA-NJ 14

organism: Macaca mulatta

source_name: Vastus lateralis muscle_Middle-aged

growth_protocol: Experimental protocol: Young (6.00±0.00 y), middle-aged (16.48±0.31 y), and old (26.83±0.22 y) male skeletal muscle samples from rhesus monkeys (Macaca mulatta) were used in this study. Rhesus monkeys were housed continuously at the NIH Animal Center, Poolesville, MD. Monkeys were housed individually in standard nonhuman primate caging on a 12h light/12h dark cycle, room temperature 25.5 ± 0.5 °C, humidity at 60 ± 20%. Monkeys were maintained on standard monkey chow (TestDiet® #5038 Purina Mills, Richmond, IN), and received 2 meals per day at estimated ad libitum levels throughout the study and water was always available. Monkeys were monitored minimally 3 times daily by trained animal care staff. All animal procedures for this study were reviewed and approved by the Animal Care and Use Committee (ACUC) at the Biomedical Research Center (NIA/NIH).

extract_protocol: RNA was isolated from skeletal muscle using Trizol Reagent (Invitrogen, Carlsbad, CA) following the manufacturer’s instructions

treatment_protocol: Biopsies of the vastus lateralis were obtained from the monkeys from ongoing NIA studies, and flash frozen in liquid nitrogen and stored at -80oC until assayed.

value: Middle-aged


title: T4_BF_8_9_14_U

data_processing:
Illumina reads were mapped to the Streptococcus pneumoniae TIGR4 genome using Bowtie v. 0.12.7
The mapped reads were counted using HTSeq and normalized and then the differential expression of each gene for one sample compared to another was determined using DESeq v. 1.5.24, and statistical analyses were performed using R-2.15.2, and was represented as the log2 of the fold-change values.
RPKMs for each gene were calculated for each sample.
Genome_build: NC_003028.3
Supplementary_files_format_and_content: Tab-delimited text files include RPKMs

source_name: Streptococcus pneumoniae cells, media-grown biofilm sample

growth_protocol: Pneumococci were grown in Todd Hewitt Broth (THB) (Acumedia, Neogen) with 0.5% yeast extract (THY) at 37°C in 5% CO2 until reaching mid-exponential phase [Optical density, O.D.620nm = 0.5; ~1.0 x 108 colony forming units (CFU)/mL] and serially diluted in sterile phosphate buffered saline (PBS). Blood was collected from anesthetized mice retro-orbitally and transferred to heparin-coated collection tubes. Following euthanasia, hearts were surgically excised and washed in PBS to remove blood. Isolated hearts were homogenized in 5ml of PBS followed by filtration of the homogenate through a 40µm cell strainer. Paired blood (BIP) and strained heart (HIP) samples were flash-frozen at -80° C in working aliquots with 10% glycerol.

extract_protocol:
For HIP-RNA, hearts were excised from HIP-infected mice (n=3) when deemed moribund. Hearts were rinsed, diced, fragments washed with ice cold PBS, and the fragments homogenized in RNAprotect bacteria reagent (Qiagen). For BIP-RNA, 10 mice pre-depleted for neutrophils using anti-Ly6G antibody (BioXCell, clone RB6-8C5), were infected with TIGR4 and blood was collected in RNAprotect bacteria reagent (Qiagen) when the mice were deemed moribund such that blood from 5 mice were pooled as one sample (n=2 BIP samples). These heart homogenates were stored at -80°C. On the day of total RNA isolation from heart homogenates, the samples were thawed and spun down to discard the supernatant. The pellets were further homogenized in 600 ?L RLT with B-ME buffer using a motorized mortar for 30 seconds. The re-homogenized samples were then disaggregated in a Qiashredder followed by RNA extraction with the RNeasy Micro Kit (Qiagen) with DNase treatment on column and in solution. The isolated RNA was quantitated using Nanodrop and Bioanalyzer. Samples were then depleted of rRNAs using the Ribo-Zero rRNA Removal Kit for Gram-positive bacteria and human/mouse/rat (Illumina, San Diego, CA). For the in vitro biofilm and planktonic samples: planktonic mid-log phase (OD620nm = 0.5) TIGR4 grown in THB were used to seed continuous once-flow through biofilm reactors. Biofilms were allowed to grow for 48 hours prior to collection of bacteria. RNA was isolated from the planktonic seed cultures and their respective biofilms. In total, 3 independent biological samples were collected with planktonic and biofilm RNAs from each experiment paired. Total RNA was extracted from each replicate separately using enzymatic lysis of pneumococcal cells (10 ?L mutanolysin, 20 ?L proteinase K, 15 ?L lysozyme, and 55 ?L TE) followed by RNA extraction using the same protocol mentioned above. Blood samples were isolated in a similar manner as biofilm and planktonic samples, including enzymatic lysis, except that cells were first disaggregated with the Qiashredder like for the heart samples.
RNA libraries were prepared for sequencing using standard Illumina protocols.

value: biofilm sample


title: T4_PK_8_6_14_U

source_name: Streptococcus pneumoniae cells, media-grown planktonic sample

value: planktonic sample


title: IR1

source_name: Streptococcus pneumoniae cells, infected mouse heart sample

value: infected mouse heart


title: Extra_lymph_nodes_GIT2KO_HO_replicate_2

value: extra lymph nodes


title: T4_PK_7_24_14_T


title: T4_PK_8_9_14_U


title: IR4


title: T4_BF_8_6_14_U


title: T4_BF_7_25_14_T


title: IR3


title: Extra_lymph_nodes_GIT2KO_HO_replicate_3


title: Extra_lymph_nodes_GIT2KO_HO_replicate_1


title: HAEC_Control_Replicate_2

status: Public on Aug 11 2017

scan_protocol: Arrays were scanned at a resolution of 0.54 μm using the Illumina iScan scanner.

growth_protocol: Primary human aortic endothelial cells (HAEC) were grown in EMB-2 supplemented with the EGM-2 SingleQuot Kit (Lonza; Walkersville, MD),  and were transfected with pre-miR miRNA precursors for hsa-miR-103a-2-5p, miR-585-5p, or scrambled negative control (Life Technologies) using Lipofectamine 2000 (Life Technologies).

extract_protocol: RNA was extracted using Trizol (Trizol Reagent, Invitrogen) according to manufacturer’s instructions. RNA was evaluated by Nanodrop and RNA concentration and integrity was assessed by Agilent Bioanalyzer RNA 6000 Chip (Agilent, Santa Clara, CA).

value: Primary human aortic endothelial cells (HAECs)

tag: transfected pre-mir

value: Scrambled, negative control


title: HAEC_miR-103a_Replicate_3


title: HAEC_Control_Replicate_3


title: HAEC_miR-585_Replicate_1


title: Pleural_LTS_7day_G4_rep4

status: Public on Jun 08 2017

city: River Forest

first: Robert

middle: J

last: Calin-Jageman

state: IL

address: 7900 West Division

department: Psychology

institute: Dominican University

description: Z73: Animals 1059,1060

hyb_protocol: We used the Aplysia Tellabs Array (ATA: GEO: GPL18666) to characterize changes in gene expression persisting 7 days after long-term sensitization training.  The array was designed using the UniGene clustering of Aplysia ESTs (build 9, July 2011, ftp://ftp.ncbi.nih.gov/repository/UniGene/Aplysia_californica/) and includes 2-3 probes designed for each of the 24,702 distinct EST clusters identified by Unigene, 15 Aplysia mRNAs deposited in GenBank between the Unigene build and the design of the array, and 1,432 orphaned probes from two previous Aplysia microarray designs (the Aplysia Discovery Array (GEO:GPL3635) and the updated Aplysia Annotated Array (GEO: GPL13815/GPL17112)).  Thus, the ATA design includes 26,149 distinct probes representing all known sources of Aplysia californica ESTs and mRNAs at the time of design (January 2012).   Based on estimates from previous microarray designs (Moroz et al. 2006), the ATA should cover >50-60% of all neurally-expressed transcripts.  Complete details on the ATA design are in Herdegen et al. (Herdegen et al. 2014b).  Microarray processing was completed by Mogene Inc. (St. Louis, MO).  A two-color approach was used with each array hybridized to a sample from a trained or untrained animal.  In half of cases, trained samples were hybridized with Cy3 and controls to Cy5; the other half we dye-swapped.    Sample integrity was determined by Bioanalyzer RNA 6000, Pico total RNA protocol.  300ng of total RNA was amplified and labeled with Cy3 or Cy5 using the Agilent Quick Amp Two-Color Labeling Kit. Dye incorporation and yield was determined by Nanodrop.  Samples were hybridized to the microarray slide at 65C and 10rpm for 17 hours.  Slides were scanned on an Agilent C scanner at 3µm resolution.  Data was extracted using Agilent Feature Extraction software, v. 11.5.  All labeling and post-labeling processing was carried out in an ozone regulated environment, monitored at < 5ppb.

scan_protocol: Slides were scanned on an Agilent C scanner at 3um resolution.  Data was extracted using Agilent Feature Extraction software, v. 11.5.

data_processing: Microarray data was analyzed using limma (Smyth 2005; Ritchie et al. 2015a) from the Bioconductor suite of tools (Gentleman et al. 2004) for R (Ihaka and Gentleman 1996).   Median expression values were analyzed (Zahurak et al. 2007).  These were corrected for background using the normexp+offset algorithm recommended for Agilent microarrays by Ritchie et al. (Ritchie et al. 2007).  An offset of 30 was selected based on inspection of MA Plots (Supplemental Figure 2).  Expression was then normalized using the loess function (Smyth and Speed 2003).  Where multiple probes were used to measure the same EST or mRNA, these were averaged.  Finally, trained and control expression were compared using an empirical Bayes-moderated t-test (Smyth 2004).  Statistical significance was calculated using Benjamini-Hochberg correction for multiple comparisons to maintain a 5% overall false-discovery rate (Benjamini and Hochberg 1995).  We used the treat function from limma (McCarthy and Smyth 2009) to conduct a stringent test for significant regulation.  Specifically, rather than use a null hypothesis of no regulation, we tested for regulation statistically distinguishable from at least a 10% change in expression in either direction.  We have previously found that using this type of high-stringency criterion yields very strong predictive validity in independent qPCR (Herdegen et al. 2014b; Holmes et al. 2014).   For this analyiss, we first tested for regulation in the set of 1198 transcripts known to be regulated 24 hours after training (Conte et al., 2017).  Then we tested for unique regulation in the remaining transcripts.  We also explored the completeness of the gene list using the approach developed by Langaas et al. (2005), which is implemented in limma via the propTrueNull function (Ritchie et al. 2015b).  We also explored the degree of relationship between the regulation observed 7 days after training with what we have previoulsy observed 1 day after training (Conte et al., 2017).  Specifically, we examined both the correlation between raw log-fold-change scores and the correlation once these scores are adjusted for potential measurement error using the genuine association of gene-expression profiles (genas) function in limma (Ritchie et al. 2015b)

organism: Aplysia californica

source_name: control

label_protocol: A two-color approach was used with each array hybridized to a trained (Cy5) or untrained (Cy3) sample.  Sample integrity was determined by Bioanalyzer RNA 6000, Pico total RNA protocol.  300ng of total RNA was amplified and labeled with Cy3 or Cy5 using the Agilent Quick Amp Two-Color Labeling Kit. Dye incorporation and yield was determined by Nanodrop.

growth_protocol: Animals (75-125g) were obtained from the RSMAS National Resource for Aplysia (Miami, FL) and maintained at 16o C in one of two 90-gallon aquariums with continuously circulating artificial sea water (Instant Ocean, Aquarium Systems Inc.).  Animals were separately housed in rectangular colanders (11.5cm wide, 16.5cm long, 7.3cm deep), fed dried seaweed twice a week, and maintained on a 12 hr light-dark cycle.  2 days prior to any experimental testing, animals were fed to satiation and then food deprived until after the 1-day post-tests (Levenson, Byrne, & Eskin, 1999; Wainwright, Zhang, Byrne, & Cleary, 2002).  Food deprivation was resumed the day before the 7-day post-tests.  To control for batch/shipment effects, animals from at least 2 different shipments were used for each experiment.

extract_protocol: We compared gene expression from pleural ganglia on the trained vs. untrained side of the animal.  Samples from two animals trained on opposite sides were pooled.  This was done to control for lateralized gene expression.  To analyze transcription, pleural ganglia RNA was isolated immediately after the 7-day post-tests, a time-point at which recall had fully decayed (yet it remains possible to evoke savings memory).  Briefly, animals were anesthetized with an injection of isotonic MgCl2 (50% of body weight), and an incision was then made along the ventral midline to expose the CNS.  As dissection can alter gene expression (Alberini et al. 1994), we extracted ganglia rapidly (< 5 minutes per animal) and transferred them immediately to Trizol (Invitrogen, Carlsbad CA) for homogenization.    Tissue was homogenized using the Bullet Blender (NextAdvance, Averill Park, NY) and RNA extracted using Direct-Zol Mini RNA Kit (Zymo, Irvine, CA). Quantity and quality of RNA was assessed using the NanoDrop 1000 (Thermo Scientific, Wilmington, DE).

treatment_protocol: A one-day long-term sensitization training protocol (Figure 1) was used, adapted from Wainwright et al. (2002) as previously described (Bonnick et al. 2012; Herdegen et al. 2014b, 2014a)  Training consisted of 4 rounds of noxious shock applied at 30 minute intervals to one side of the body with a hand-held electrode.  Each round of shock consisted of 10 pulses (60Hz biphasic) of 500ms duration at a rate of 1hz and an amplitude of 90mA.  During the course of each shock, the stimulating electrode was slowly moved from anterior (just behind neck) to posterior (just in front of tail) and back to cover nearly the entire surface of that side of the body.  Side of training was counterbalanced.  One of the advantages of this training protocol is that both the induction and expression of sensitization is restricted to one side of the body (Antzoulatos and Byrne 2007; Antzoulatos et al. 2006; Cleary et al. 1998; Scholz and Byrne 1987; Wainwright et al. 2002), enabling each animal to serve as its own control (trained vs. untrained sides).'

value: pleural ganglia

source_name: trained


title: Pleural_LTS_7day_G1_rep1

description: Z49: Animals 997,998


title: HAEC_miR-103a_Replicate_2


title: HAEC_miR-585_Replicate_3


title: Pleural_LTS_7day_G5_rep5

description: Z74: Animals 1037,1038


title: Pleural_LTS_7day_G8_rep8

description: Z78: Animals 1045,1046


title: HAEC_miR-585_Replicate_2


title: HAEC_miR-103a_Replicate_1


title: Pleural_LTS_7day_G3_rep3

description: Z66: Animals 1023,1024


title: Pleural_LTS_7day_G2_rep2

description: Z64: Animals 1019,1020


title: Pleural_LTS_7day_G7_rep7

description: Z77: Animals 1043,1044


title: Pleural_LTS_7day_G6_rep6

description: Z76: Animals 1041,1042


title: HAEC_Control_Replicate_1


title: HepG2_Ctr_ NQ01 IP_replicate_2

status: Public on May 22 2017

description:
RIP
CT-14-NQO1

scan_protocol: Arrays were scanned at a resolution of 0.53 μm using the Illumina iScan scanner.

data_processing: Data was extracted using the Illumina GenomeStudio software V2011.1, Gene expression module 1.9.0. Any spots at or below the background were filtered out using an Illumina detection Pvalue of 0.02 and above. The natural log of all remaining scores were used to find the avg and std of each array and the z-score normalization was calculated and presented in the data table.  Z-score = (raw value - avg)/std.  Complete data including detection scores is supplied.

source_name: HepG2_untreated_NQO1 IP_Rep-2

growth_protocol: Human HepG2  hepatocarcinoma cells were cultured in DMEM supplemented with Glutamax, 10% FBS, and penicillin/streptomycin (all from Invitrogen) under standard cell culture conditions.

extract_protocol: Cytoplasmic cell lysates were prepared in polysome lysis buffer (PLB) [100 mM KCl, 5 mM MgCl2, 10 mM HEPES, pH 7.0, 0.5% Nonidet P-40, 1 mM dithiothreitol (DTT)] containing 100 U/ml RNaseOUT (Invitrogen) and a protease inhibitor cocktail (Roche).  For microarray analysis, 3 mg of lysate were incubated (2 h, 4°C) with 100 ml of a 50% (v/v) suspension of protein-A Sepharose beads precoated with 20 mg each of mouse anti-NQO1 or mouse IgG (Santa Cruz Biotechnology). RNA was isolated from cell extracts or from RIP samples using TRIzol (Thermo Fisher Scientific) following the manufacturer’s procedures. RNA quality and quantity was assessed using the RNA 6000 Nano Chip on an Agilent Bioanalyzer (Agilent, Wilmington, DE).

treatment_protocol: Cells were treated either with regular culture medium (Control) or medium containing 10 microM β-lapachone (β-lap) for 30 minutes.

value: Liver

tag: tumor

value: Hepatocarcinoma

tag: cell line

value: Control

tag: immunoprecipitation

value: NQO1 IP

tag: antibody


title: HepG2_b-lap_ NQ01 IP_replicate_2

description:
RIP
Beta-lap-22-NQO1

source_name: HepG2+beta-lap_NQ01 IP_Rep-2

value: 10 microM b-lapachone (b-lap)


title: HepG2_Ctr_ NQ01 IP_replicate_4

description:
RIP
CT-16-NQO1

source_name: HepG2_untreated_NQO1 IP_Rep-4


title: HepG2_Ctr_ NQ01 IP_replicate_1

description:
RIP
CT-13-NQO1

source_name: HepG2_untreated_NQO1 IP_Rep-1


title: HepG2_Ctr_ NQ01 IP_replicate_3

description:
RIP
CT-15-NQO1

source_name: HepG2_untreated_NQO1 IP_Rep-3


title: HepG2_b-lap_ NQ01 IP_replicate_1

description:
RIP
Beta-lap-21-NQO1

source_name: HepG2+beta-lap_NQ01 IP_Rep-1


title: HepG2_b-lap_IgG IP_replicate_2

description:
RIP
Beta-lap-10-IgG

source_name: HepG2+beta-lap _IgG IP_Rep-2

value: IgG IP

value: Mouse IgG cat# sc-2025 lot# K1814


title: HepG2_b-lap_ NQ01 IP_replicate_3

description:
RIP
Beta-lap-23-NQO1

source_name: HepG2+beta-lap_NQ01 IP_Rep-3


title: HepG2_b-lap_ NQ01 IP_replicate_4

description:
RIP
Beta-lap-24-NQO1

source_name: HepG2+beta-lap_NQ01 IP_Rep-4


title: HepG2_b-lap_IgG IP_replicate_4

description:
RIP
Beta-lap-12-IgG

source_name: HepG2+beta-lap _IgG IP_Rep-4


title: Hypothalamus_WT_Rep2

status: Public on May 17 2017

hyb_protocol: Standard Illumina protocol. In short, a total of 0.75 μg of biotin-labeled cRNA was hybridized at 58°C for 16 hours to Illumina's Sentrix Mouse Ref-8 v3 Expression Bead Chips (Illumina, San Diego, CA). Each BeadChip has more than 47,000 probes derived from the National Center for Biotechnology Information Reference Sequence (NCBI) RefSeq Release 38 (November 7, 2009) and other sources. The arrays were washed, blocked and the biotin labeled cRNA was detected by staining with streptavidin-Cy3.

scan_protocol: Arrays were scanned at a resolution of 0.8um using the Illumina Beadstation 500 X

data_processing:
Data was extracted using the Illumina GenomeStudio software V2011.1, Gene expression module 1.9.0. Any spots at or below the background were filtered out using an Illumina detection Pvalue of 0.02 and above. The natural log of all remaining scores were used to find the avg and std of each array and the z-score normalization was calculated and presented below.  Z-score = (raw value - avg)/std.  Complete data including detection scores is supplied.
Z_VALUE = Z transformation of the natural log of the raw intensity values
Detection_Pval = Detection Pvalue from Illumina GenomeStudio software V2011.1, Gene expression module 1.9.0.

extract_protocol: Dissected mouse brain regions were first homogenized using a Bead Beater (Bio-Spec, Bartlesville, OK) followed by RNA purification using the RNEasy Mini Kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions. RNA quality and quantity was assessed using the RNA 6000 Nano Chip on an Agilent Bioanalyzer (Agilent, Wilmington, DE).

tag: strain background

value: WT

value: male

tag: age

value: 4-5 months

value: Brain

tag: brain region

value: Hypothalamus


title: Hippocampus_T1R3KO_Rep3

value: T1R3 knockout (T1R3KO)

tag: tissue

value: Hippocampus


title: Hypothalamus_T1R3KO_Rep2

tag: gender


title: Hippocampus_WT_Rep1


title: Hypothalamus_T1R3KO_Rep3


title: Cortex_WT_Rep1

value: Cortex


title: HepG2_Ctr_ IgG IP_replicate_4

description:
RIP
CT-4-IgG

source_name: HepG2_untreated_IgG IP_Rep-4

tag: treatment


title: Hypothalamus_WT_Rep1


title: HepG2_Ctr_ IgG IP_replicate_3

description:
RIP
CT-3-IgG

source_name: HepG2_untreated_IgG IP_Rep-3


title: HepG2_b-lap_ IgG IP_replicate_1

description:
RIP
Beta-lap-9-IgG

source_name: HepG2+beta-lap _IgG IP_Rep-1


title: HepG2_Ctr_ IgG IP_replicate_1

description:
RIP
CT-1-IgG

source_name: HepG2_untreated_IgG IP_Rep-1


title: Hypothalamus_T1R3KO_Rep1


title: HepG2_Ctr_ IgG IP_replicate_2

description:
RIP
CT-2-IgG

source_name: HepG2_untreated_IgG IP_Rep-2


title: Hypothalamus_WT_Rep3


title: Hippocampus_T1R3KO_Rep2


title: Hippocampus_WT_Rep2


title: Hippocampus_WT_Rep3


title: Cortex_WT_Rep2


title: S22_GM_RPAD_2_S19

status: Public on May 03 2017

description: Circular RNA

data_processing:
The reads were cleaned usning cutadapt program
The tags were aligned using Tophat and Tophat fusion (v2.1.0) with --GTF option using hg19 Ensembl v82 annotation database and mm9 Ensembl v67 annotation database
Cufflinks (v2.2.1) was used to assemble the transcripts with GTF-guide and minimum of 5 reads supporting the transcript assembly
CIRCexplorer (v1.1.10) was used to find circRNAs
Genome_build: hg19 and mm9
Supplementary_files_format_and_content: The processed data file contain information about the circRNA and the junctional read counts in a tab-delimited TXT format.

source_name: Myoblast

growth_protocol: Human cervical carcinoma HeLa cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Invitrogen) containing 10% fetal bovine serum (FBS, Gibco) and antibiotics (Life Technologies).  Mouse C2C12 myoblasts were cultured in DMEM supplemented with 20% FBS and antibiotics.  Both Hela and C2C12 cells were maintained at 37°C in a humidified atmosphere of 95% air, 5% CO2.

extract_protocol:
Total RNA from cultured cells was isolated using the miRNeasy Mini Kit (#217004, Qiagen Inc.) following the manufacturer’s protocol. For the RPAD samples, a 20-µl reaction was set with 2 µg of Hela RNA, 20 units of RiboLock RNase inhibitor (Thermo Fisher Scientific), 1× RNase R buffer and 20 units of RNase R (RNR07250, Epicentre), and incubated for 30 min at 37°C.  The RNA from control and RNase R-treated samples were isolated using miRNeasy Mini Kit following the manufacturer’s instructions. The RNase R treated sample was treated with PolyA polymerase to ass polyA tail to linear RNAs followed by incubation with Oligo-dT Dynabeads from Poly(A)Purist™ MAG Kit (AM1922) and the supernatant was collected for RNA isolation.  The miRNeasy Mini Kit was used to prepare RNA from the control and RNase R-polyadenylation and poly(A)+ RNA depletion (RPAD) samples.
After isolation of total RNA and RPAD-prepared RNA using miRNeasy Mini Kit, ribosomal (r)RNA was removed by rRNA Depletion Nano kit (Qiagen), whereupon cDNA was prepared and amplified using the Ovation RNA-Seq System V2 (NuGEN) kit following the manufacturer’s instructions.  The amplified cDNA was fragmented using a Bioruptor (Diagenode) followed by ligation of adaptors using TruSeq ChiP Sample Preparation kit (Illumina, San Diego, CA) and DNA fragments ranging 300-350 bp were isolated from a 2.5% agarose gel and subjected to 17 cycles of PCR amplification.  A Bioanalyzer 2100 instrument was used to analyze the quality of the cDNA libraries, which were sequenced using an Illumina HiSeq2500 instrument.

treatment_protocol: RPAD (RNase R digestion, polyadenylation, and poly(A)+ RNA depletion) yields almost-pure circRNAs


title: S19_GM_1_S13

description: Circular RNA


title: S20_GM_2_S14

description: Circular RNA


title: S14_Hela_RPAD_1_S22

description: Circular RNA

source_name: Cervical carcinoma

value: HeLa


title: HepG2_b-lap_IgG IP_replicate_3

description:
RIP
Beta-lap-11-IgG

source_name: HepG2+beta-lap _IgG IP_Rep-3


title: S13_Hela_1_S17

description: Circular RNA


title: Cortex_WT_Rep3


title: RNAseq_kidney_KS_tamoxifen_4

status: Public on Apr 26 2017

city: Chapel Hill

first: William

middle: Y.

last: Kim

state: NC

address: 450 West Dr.

department: Medicine and Genetics

institute: University of North Carolina at Chapel Hill

data_processing:
75b paired-end reads were sequenced on a NextSeq 500 Desktop Sequencer using a high output flow cell kit (Illumina), yielding an average of over 28M reads per sample.
QC-passed reads were aligned to the mouse reference genome (mm9) using MapSplice
The alignment profile was determined by Picard Tools v1.64 (http://broadinstitute.github.io/picard/).
Aligned reads were sorted and indexed using SAMtools and translated to transcriptome coordinates then filtered for indels, large inserts, and zero mapping quality using UBU v1.0 (https://github.com/mozack/ubu).
Transcript abundance estimates for each sample were performed using RSEM, an expectation-maximization algorithm using the UCSC knownGene transcript and gene definitions.
Raw RSEM read counts for all RNAseq samples were normalized to the overall upper quartile
Genome_build: mm9
Supplementary_files_format_and_content: Tab-delimited text files include RSEM values for each sample

source_name: kidney

extract_protocol:
RNA was extracted with Qiagen Rneasy Kit
Libraries were prepared using TruSeq Stranded mRNA Sample Prep Kit (Illumina)

value: kidney

tag: kidney region

value: cortex

tag: genotype

value: tamoxifen

tag: genotype abbreviations

value: KS

tag: background strain

value: mixed


title: Cortex_T1R3KO_Rep1

tag: genotype/variation


title: Cortex_T1R3KO_Rep2


title: RNAseq_kidney_KS_tamoxifen_2


title: RNAseq_kidney_Ctrl_vehicle_3

value: vehicle

value: Ctrl


title: RNAseq_kidney_Ctrl_vehicle_1


title: RNAseq_kidney_Ctrl_vehicle_2


title: KT-J22-KT3_S17

status: Public on Apr 12 2017

extract_protocol:
RNA was extracted with Qiagen Rneasy Kit
Libraries were prepared using TruSeq Stranded mRNA Sample Prep Kit (Illumina)
RNAseq-Paired-end

value: Wild Type


title: KT-J20-KT1_S15

value: WT


title: Hippocampus_T1R3KO_Rep1

value: male


title: B6-J19-B62_S14


title: KT-J21-KT2_S16


title: Cortex_T1R3KO_Rep3

value: 4-5 months


title: B6-J18-B61_S13


title: VM-J17-2459_S12

value: Vhl-/-; MYC +

value: VM

value: maintained on dox


title: VM-J16-3055_S11


title: VM-J15-3115_S10


title: VIM-J13-3130_S8

value: VIM


title: VIM-J7-3237_S5


title: VIM-J10-3233_S6


title: M-J6-2983_S4

value: MYC+

value: M


title: M-J1-2917_S1


title: M-J5-2983_S3


title: J54-VIM-no-dox4_S16

value: withdrew dox


title: J53-VIM-no-dox3_S15


title: RNAseq_kidney_KS_tamoxifen_1

value: kidney

value: cortex


title: RNAseq_kidney_KS_tamoxifen_3

value: tamoxifen

value: KS


title: J52-VIM-no-dox2_S14


title: RNAseq_kidney_Ctrl_vehicle_4

value: vehicle

value: mixed


title: J50-VIM-dox4_S12


title: J35-VM-no-dox1_S5


title: J37-VM-no-dox3_S7


title: J48-VIM-dox2_S10


title: J34-VM-dox4_S4


title: J36-VM-no-dox2_S6


title: J38-VM-no-dox4_S8


title: J32-VM-dox2_S2


title: J31-VM-dox1_S1


title: J62-M2983-no-dox_S7


title: J63-M2983-no-dox_S8


title: J61-M2983-no-dox_S6


title: J58-M2983-dox_S4


title: J57-M2983-dox_S3


title: J56-M2983-dox_S2


title: J60-M2983-no-dox_S5


title: J51-VIM-no-dox1_S13


title: J55-M2983-dox_S1


title: Hepatocyte-Like Cell - 4105B

status: Public on Apr 01 2017

city: Cambridge

first: Curtis

middle: Robert

last: Warren

state: Massachusetts

address: 7 Divinity Ave

department: Stem Cell And Regenerative Biology

institute: Harvard University

data_processing:
Assembly - Bowtie 2 (TopHat2.0.13)
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence, then mapped to Homo_sapiens/Ensembl/GRCh37 whole genome sequence using  TopHat2.0.13 with parameters "tophat -p 16 -g 1 -N 2 -r 50 --mate-std-dev 70 --no-coverage-search -o"
Fragments per kilobase of transcript per million mapped reads  (FPKM) were calculated using cufflinks/2.2.1 with gene/transcript annotation ("Ensembl_GRCh37.v74.gtf") with parameters "-G Homo_sapiens.GRCh37.74.gtf --total-hits-norm --no-update-check --library-type fr-unstranded "
rseqc/v2.6.2 for generating mapping statistics metrics from accepted.bam files
Genome_build: NCBI v37 Homo sapiens Reference Genome (hg19)
Supplementary_files_format_and_content: tab-delimited text file include FPKM values for each Sample

source_name: Peripheral Blood Mononuclear Cells

growth_protocol: Warren et al Cell Stem Cell 2016

extract_protocol:
Warren et al Cell Stem Cell 2016
Warren et al Cell Stem Cell 2016

treatment_protocol: Warren et al Cell Stem Cell 2016

value: Peripheral Blood Mononuclear Cells

tag: cell type

value: Hepatocyte-Like Cell

tag: 1p13 genotype

value: Minor/Minor


title: Hepatocyte-Like Cell - 4105D


title: Hepatocyte-Like Cell - 2633B


title: Hepatocyte-Like Cell - 25727B


title: Hepatocyte-Like Cell - 2633D


title: Hepatocyte-Like Cell - 25727G


title: Hepatocyte-Like Cell - 25642C


title: J49-VIM-dox3_S11


title: J47-VIM-dox1_S9


title: J33-VM-dox3_S3


title: Hepatocyte-Like Cell - 25642B


title: Hepatocyte-Like Cell - 1998E


title: Hepatocyte-Like Cell - 24472D


title: Hepatocyte-Like Cell - 16939A


title: Hepatocyte-Like Cell - 24472K


title: Hepatocyte-Like Cell - 16939B


title: Hepatocyte-Like Cell - 1998G


title: Hepatocyte-Like Cell - 22512A


title: Hepatocyte-Like Cell - 22512D


title: Hepatocyte-Like Cell - 16126E


title: Hepatocyte-Like Cell - 16126C


title: Hepatocyte-Like Cell - 14685A


title: Hepatocyte-Like Cell - 14157C


title: Hepatocyte-Like Cell - 14157A


title: Hepatocyte-Like Cell - 14685B


title: Hepatocyte-Like Cell - 13352A


title: Hepatocyte-Like Cell - 1296E


title: Hepatocyte-Like Cell - 12888B


title: Hepatocyte-Like Cell - 1296C


title: Hepatocyte-Like Cell - 12888A


title: Hepatocyte-Like Cell - 1236B


title: Hepatocyte-Like Cell - 12022C


title: Hepatocyte-Like Cell - 12022B


title: Hepatocyte-Like Cell - 10950C


title: Hepatocyte-Like Cell - 10950A


title: Hepatocyte-Like Cell - 9462B

value: Major/Major


title: Hepatocyte-Like Cell - 4756A


title: Hepatocyte-Like Cell - 4756B


title: Hepatocyte-Like Cell - 554D


title: Hepatocyte-Like Cell - 554A


title: Hepatocyte-Like Cell - 921B


title: Hepatocyte-Like Cell - 521E


title: Hepatocyte-Like Cell - 9462A


title: Hepatocyte-Like Cell - 4064B


title: Hepatocyte-Like Cell - 4064A


title: Hepatocyte-Like Cell - 521C


title: Hepatocyte-Like Cell - 921C


title: Hepatocyte-Like Cell - 3726A


title: Hepatocyte-Like Cell - 3726B


title: Hepatocyte-Like Cell - 13352E


title: Hepatocyte-Like Cell - 2945A


title: Hepatocyte-Like Cell - 2098B


title: Hepatocyte-Like Cell - 2098A


title: Hepatocyte-Like Cell - 18961A


title: Hepatocyte-Like Cell - 18719D


title: Hepatocyte-Like Cell - 1236C


title: Hepatocyte-Like Cell - 18719B


title: Hepatocyte-Like Cell - 1673F


title: Hepatocyte-Like Cell - 1673B


title: Hepatocyte-Like Cell - 1660F


title: Hepatocyte-Like Cell - 1660E


title: Hepatocyte-Like Cell - 1649B


title: Liver differentiation day 21

status: Public on Apr 28 2017

city: Shenzhen

first: Andrew

middle: P

last: Hutchins

address: 1088 Xueyuan Rd

department: Bioinformatics

country: China

institute: Southern University of Science and Technology

description: Presumed hepatocyte-like

data_processing:
rsem v1.2.17/bowtie2 v2.2.0 was used to align to the ensembl transcriptome (mm10, v76)
transcripts with more than 25 sequence tags in 2 or more timepoints were kept for further analysis
mapping tag counts were GC normalised using EDASeq v2.0.0
Genome_build: hg19
Supplementary_files_format_and_content: tab delimeted text file containing ensembl transcript accession, gene name and normalised abundance estimates for each transcript

source_name: Differentiated hESCs

growth_protocol: Undifferentiated human H1 ES cells were maintained in monolayer culture on Matrigel (BD Biosciences, 354277) in mTeSR1 medium (Stemcell Technologies, 05850) at 37°C with 5% CO2. Cells were manually passaged at 1:4 to 1:6 split ratios every 3 to 5 days.

extract_protocol:
TRIzol
RNA libraries were prepared for sequencing using standard Illumina protocols

treatment_protocol: For hepatic differentiation, we established a serum-free protocol based on previously described protocols with minor modifications (Si-Tayeb et al., 2010; Song et al., 2009). Briefly, cells were cultured for 3 days in RPMI/B27 medium (Insulin minus, Gibco, A18956-01) supplemented with 100 ng/ml Activin A (Peprotech, A120-14E), followed by 4 days with 20 ng/ml BMP2 (Peprotech, 120-02)and 30 ng/ml FGF-4 (Peprotech, 100-31) in RPMI/B27(complete with Insulin, Gibco,17504-044) medium, then 6 days with 20 ng/ml HGF (Peprotech, 100-39) and KGF (Peprotech, 100-19) in RPMI/B27 (complete with Insulin), then 8 days with 20 ng/ml Oncostatin-M (R&D Systems, 295-OM/CF) in Hepatocyte Culture Media (Lonza, cc-3198) supplemented with SingleQuots (without EGF).

tag: day

value: OSM

tag: presumed cell type

value: Hepatocyte-like


title: Hepatocyte-Like Cell - 1441G

tag: cell line


title: Hepatocyte-Like Cell - 1293A


title: Hepatocyte-Like Cell - 12569B


title: Hepatocyte-Like Cell - 1293B


title: Hepatocyte-Like Cell - 2945B


title: Hepatocyte-Like Cell - 1649A


title: Hepatocyte-Like Cell - 18961C


title: Hepatocyte-Like Cell - 1441F


title: Hepatocyte-Like Cell - 12569A


title: S21_GM_RPAD_1_S18

description: Circular RNA


title: 120714_PBMC_28

status: Public on Mar 01 2017

city: New Haven

first: Hailong

last: Meng

state: CT

address: 300 George Str

institute: Yale University

hyb_protocol: Standard Illumina hybridization protocol

scan_protocol: Standard Illumina scanning protocol

data_processing: The data were normalised using quantile normalisation with lumi in R

source_name: PBMC

label_protocol: Preparation of cDNA, cRNA, and labeling were carried out by the Yale Center for Genomic Analysis using the standard Illumina protocols.   Hybridization buffer from the BeadChip kit (Illumina) was mixed with 1500 ng of biotin-labeled cRNA, heated to 65°C for 5 minutes, and then loaded onto the BeadChip.  The BeadChips were sealed in a hybridization chamber and placed in an oven at 58°C with a rocker for 16-20 hours.  After the hybridization, the BeadChips were washed and stained in a series of washes and stains as outlined in the Illumina protocol.  The BeadChips were scanned on the Illumina Iscan.

extract_protocol: Total RNA from PBMC, T Cell, B Cell samples was extracted using the RNeasy mini kit (Qiagen, CA)

tag: blood draw date

tag: age group

value: OLD

tag: phenotype

value: R


title: 120712_PBMC_28

value: YOUNG


title: 120714_PBMC_0


title: 120714_PBMC_9


title: Pleural_LTS_24hr_G8_rep8

status: Public on Mar 03 2017

description: Z39: Animals 985, 988

hyb_protocol: We used the Aplysia Tellabs Array (ATA: GEO: GPL18666) to characterize changes in gene expression due to long-term habituation training.  The array was designed using the UniGene clustering of Aplysia ESTs (build 9, July 2011, ftp://ftp.ncbi.nih.gov/repository/UniGene/Aplysia_californica/) and includes 2-3 probes designed for each of the 24,702 distinct EST clusters identified by Unigene, 15 Aplysia mRNAs deposited in GenBank between the Unigene build and the design of the array, and 1,432 orphaned probes from two previous Aplysia microarray designs (the Aplysia Discovery Array (GEO:GPL3635) and the updated Aplysia Annotated Array (GEO: GPL13815/GPL17112)).  Thus, the ATA design includes 26,149 distinct probes representing all known sources of Aplysia californica ESTs and mRNAs at the time of design (January 2012).   Based on estimates from previous microarray designs (Moroz et al. 2006), the ATA should cover >50-60% of all neurally-expressed transcripts.  Complete details on the ATA design are in Herdegen et al. (Herdegen et al. 2014b).  Microarray processing was completed by Mogene Inc. (St. Louis, MO).  A two-color approach was used with each array hybridized to a sample from a trained or untrained animal.  In half of cases, trained samples were hybridized with Cy3 and controls to Cy5; the other half we dye-swapped.    Sample integrity was determined by Bioanalyzer RNA 6000, Pico total RNA protocol.  300ng of total RNA was amplified and labeled with Cy3 or Cy5 using the Agilent Quick Amp Two-Color Labeling Kit. Dye incorporation and yield was determined by Nanodrop.  Samples were hybridized to the microarray slide at 65C and 10rpm for 17 hours.  Slides were scanned on an Agilent C scanner at 3µm resolution.  Data was extracted using Agilent Feature Extraction software, v. 11.5.  All labeling and post-labeling processing was carried out in an ozone regulated environment, monitored at < 5ppb.

data_processing: Microarray data was analyzed using limma (Smyth 2005; Ritchie et al. 2015a) from the Bioconductor suite of tools (Gentleman et al. 2004) for R (Ihaka and Gentleman 1996).  Our processing script for identifying differentially regulated transcripts was pre-registered and is posted on the Open Science Framework.   Median expression values were analyzed (Zahurak et al. 2007).  These were corrected for background using the normexp+offset algorithm recommended for Agilent microarrays by Ritchie et al. (Ritchie et al. 2007).  An offset of 30 was selected based on inspection of MA Plots (Supplemental Figure 2).  Expression was then normalized using the loess function (Smyth and Speed 2003).  Where multiple probes were used to measure the same EST or mRNA, these were averaged.  Finally, trained and control expression were compared using an empirical Bayes-moderated t-test (Smyth 2004).  Statistical significance was calculated using Benjamini-Hochberg correction for multiple comparisons to maintain a 5% overall false-discovery rate (Benjamini and Hochberg 1995).  We used the treat function from limma (McCarthy and Smyth 2009) to conduct a stringent test for significant regulation.  Specifically, rather than use a null hypothesis of no regulation, we tested for regulation statistically distinguishable from at least a 10% change in expression in either direction.  We have previously found that using this type of high-stringency criterion yields very strong predictive validity in independent qPCR (Herdegen et al. 2014b; Holmes et al. 2014). Expanding beyond our pre-registered analysis script we also explored the completeness of the gene list using the approach developed by Langaas et al. (2005), which is implemented in limma via the propTrueNull function (Ritchie et al. 2015b).  We also explored the degree of relationship between the regulation observed 24 hours after LTS training with our previous screen of regulation observed 1 hour after LTS training (Herdegen et al. 2014b).  We examined both the correlation between raw log-fold-change scores and the correlation once these scores are adjusted for potential measurement error using the genuine association of gene-expression profiles (genas) function in limma (Ritchie et al. 2015b)

source_name: trained

growth_protocol: Animals (75-125g) were obtained from the RSMAS National Resource for Aplysia (Miami, FL) and maintained at 16o C in one of two 90-gallon aquariums with continuously circulating artificial sea water (Instant Ocean, Aquarium Systems Inc.).  Animals were separately housed in rectangular colanders (11.5cm wide, 16.5cm long, 7.3cm deep), fed dried seaweed twice a week, and maintained on a 12 hr light-dark cycle.  2 days prior to any experimental testing, animals were fed to satiation and then food deprived for the remained of the experiment (Levenson, Byrne, & Eskin, 1999; Wainwright, Zhang, Byrne, & Cleary, 2002).  To control for batch/shipment effects, animals from at least 2 different shipments were used for each experiment.

extract_protocol: We compared gene expression from pleural ganglia on the trained vs. untrained side of the animal.  Samples from two animals trained on opposite sides were pooled.  This was done to control for lateralized gene expression.  To analyze transcription, pleural ganglia RNA was isolated immediately after the 24-hour post-tests, a time-point representing maintenance of long-term sensitization memory.  Briefly, animals were anesthetized with an injection of isotonic MgCl2 (50% of body weight), and an incision was then made along the ventral midline to expose the CNS.  As dissection can alter gene expression (Alberini et al. 1994), we extracted ganglia rapidly (< 5 minutes per animal) and transferred them immediately to Trizol (Invitrogen, Carlsbad CA) for homogenization.    Tissue was homogenized using the Bullet Blender (NextAdvance, Averill Park, NY) and RNA extracted using Direct-Zol Mini RNA Kit (Zymo, Irvine, CA). Quantity and quality of RNA was assessed using the NanoDrop 1000 (Thermo Scientific, Wilmington, DE).

value: pleural ganglia

source_name: control


title: Pleural_LTS_24hr_G6_rep6

description: Z37: Animals 980, 983


title: Pleural_LTS_24hr_G7_rep7

description: Z38: Animals 984, 987


title: 120712_PBMC_2


title: 120712_PBMC_9


title: 120706_PBMC_9


title: Pleural_LTS_24hr_G2_rep2

description: Z31: Animals 969, 972


title: 120708_PBMC_28

value: NR


title: 120711_PBMC_2


title: 120711_PBMC_9


title: Pleural_LTS_24hr_G1_rep1

description: Z30: Animals 968, 971


title: 120711_PBMC_0


title: Pleural_LTS_24hr_G3_rep3

description: Z32: Animals 970, 973


title: 120706_PBMC_28


title: 120708_PBMC_0


title: 120708_PBMC_2


title: Pleural_LTS_24hr_G5_rep5

description: Z36: Animals 979, 982


title: 120711_PBMC_28


title: 120708_PBMC_8


title: 120712_PBMC_0


title: 120706_PBMC_2


title: Pleural_LTS_24hr_G4_rep4

description: Z34: Animals 975, 977


title: 120705_PBMC_28


title: 120706_PBMC_0


title: 120714_PBMC_2


title: 120705_PBMC_0


title: 120705_PBMC_2


title: 120702_PBMC_28


title: 120702_PBMC_9


title: 120702_PBMC_0


title: 120705_PBMC_9


title: 120697_PBMC_2


title: 120697_PBMC_28


title: 120697_PBMC_0


title: 120700_PBMC_9


title: 120700_PBMC_28


title: 120702_PBMC_2


title: 120690_PBMC_4


title: 120700_PBMC_0


title: 120690_PBMC_7


title: 120697_PBMC_9


title: 120690_PBMC_0


title: 120685_PBMC_28


title: 120685_PBMC_2


title: 120683_PBMC_28


title: 120690_PBMC_28


title: 120676_PBMC_28


title: 120685_PBMC_7


title: 120683_PBMC_7


title: 120700_PBMC_2


title: 120683_PBMC_2


title: 120683_PBMC_0


title: 120676_PBMC_7


title: 120685_PBMC_0


title: 120676_PBMC_2


title: 120676_PBMC_0


title: 120674_PBMC_28


title: 120674_PBMC_7


title: 120673_PBMC_2


title: 120672_PBMC_28


title: 120673_PBMC_0


title: 120671_PBMC_28


title: 120673_PBMC_7


title: 120674_PBMC_0


title: 120672_PBMC_0


title: 120671_PBMC_7


title: 120674_PBMC_2


title: 120671_PBMC_2


title: 120660_PBMC_7


title: 120672_PBMC_2


title: 120660_PBMC_2


title: 120646_PBMC_7


title: 120646_PBMC_28


title: 120660_PBMC_28


title: 120673_PBMC_28


title: 120672_PBMC_7


title: 120671_PBMC_0


title: 120656_PBMC_2


title: 120656_PBMC_0


title: 120661_PBMC_36


title: 120656_PBMC_7


title: 120661_PBMC_0


title: 120661_PBMC_7


title: 120661_PBMC_2


title: 120656_PBMC_28


title: 120646_PBMC_2


title: 120645_PBMC_28


title: 120646_PBMC_0


title: 120645_PBMC_2


title: 120645_PBMC_0


title: 120639_PBMC_28


title: 120639_PBMC_2


title: 120645_PBMC_7


title: 120634_PBMC_8


title: 120639_PBMC_7


title: 120633_PBMC_7


title: 120637_PBMC_28


title: 120637_PBMC_2


title: 120633_PBMC_28


title: 120633_PBMC_2


title: 120634_PBMC_28


title: 120634_PBMC_2


title: 120633_PBMC_0


title: 120635_PBMC_7


title: 120631_PBMC_28


title: 120635_PBMC_0


title: 120635_PBMC_28


title: 120631_PBMC_3


title: 120629_PBMC_28


title: 120631_PBMC_7


title: 120629_PBMC_7


title: 120634_PBMC_0


title: 120637_PBMC_0


title: 120635_PBMC_2


title: 120639_PBMC_0


title: 120631_PBMC_0


title: RNA-seq 0min

city: Göttingen

first: Carina

last: Demel

address: Am Fassberg 11

country: Germany

institute: Max-Planck Institute for biophysical Chemistry

data_processing:
TT-seq and RNA-seq samples were mapped using STAR (version 2.3.0)
ChIP-seq reads were mapped with Bowtie (version 2.1.0) and duplicates were removed using PicardTools (version 1.118) MarkDuplicates.
Samtools was used to quality filter SAM files, whereby alignments with MAPQ smaller than 7 (-q 7) were skipped and only proper pairs (-f99, -f147, -f83, -f 163) were selected
PicardTools (version 1.118) CollectInsertSizeMetrics was used to determine average insert size and standard deviation with default settings: Deviations: 10.0; Minimum percentage: 0.05, Metric Accumulation Level: All reads
Genome_build: hg38 + Spikeins ERCC-00043, ERCC-00170, ERCC-00136, ERCC-00145, ERCC-00092 and ERCC-00002
Supplementary_files_format_and_content: gtf file contains the annotation used by us (available on the series record); bigwig files contain Coverage Files per sample (in case of RNAseq strand-specific antisense-corrected read coverage, in case of ChIP-seq not strand-specific, fragment coverage after duplicate removal)

source_name: T cells

growth_protocol: Cells were grown in RPMI 1640 medium (Gibco) supplemented with 10% heat-inactivated FBS (Gibco) and 1% Penicillin/Streptomycin (100x, PAA) at 37°C under 5% CO2

extract_protocol:
RNA was extracted using TRIzol (Life Technologies)
fragmentation: yes; BioRuptor Next Gen (Diagenode) at high power for one cycle of 30’’/30’’ ON/OFF
sequencing libraries were prepared with the Ovation Human Blood RNA-seq library kit (NuGEN) following the manufacturer's’ instructions.

treatment_protocol: Cells  were labeled in media for 5 min with 500 μM 4-thiouridine (4sU, Sigma-Aldrich) and harvested through centrifugation for 2 min at 3,000 rpm.

value: Jurkat cells

tag: passage


title: TT-seq 0min Rep1

description: TT-seq

extract_protocol:
RNA was extracted using TRIzol (Life Technologies), 4sU-labeled RNA was exctracted according to Dolken et al., RNA, 2008.
fragmentation: yes; BioRuptor Next Gen (Diagenode) at high power for one cycle of 30’’/30’’ ON/OFF
sequencing libraries were prepared with the Ovation Human Blood RNA-seq library kit (NuGEN) following the manufacturer's’ instructions.


title: TT-seq 0min Rep2

description: TT-seq


title: Liver_Caloric_Restriction_Replicate-5

status: Public on Feb 14 2017

description:
Total RNA including miRNA
Liver_Caloric_Restriction-5

hyb_protocol: Arrays were hybridized using the entire labeled RNA product at 55 degrees C for at least 20 hours to Agilent mouse miRNA arrays. The arrays were washed, and the bound miRNA was scanned in an Agilent scanner.

scan_protocol: Arrays were scanned at a resolution of 3um using an Agilent Array scanner, (Model G2505A)

data_processing: Data was extracted using Agilent's Feature Extractor Version 11.5.1.1, and this data was analyzed using z-score normalization. The natural log of all scores were used to find the avg and std of each array and the z-score normalization was calculated.  Z-score = (raw value - avg)/std.

label_protocol: The miRNA Complete Labeling and Hyb Kit generates fluorescently-labeled miRNA with a sample input of total RNA. This method involves the ligation of one Cyanine 3-pCp molecule to the 3' end of the RNA molecules.

growth_protocol: Three groups of one year old B6C57/J mice maintained under standard temperature and light/dark conditions.

extract_protocol: Five mice from each of these 3 groups were selected and total RNA including miRNAs was isolated using the Absolutely RNA miRNA Kit (Agilent). Quality and quantity of the RNA and miRNA was checked with the Agilent 2100 bioanalyzer using RNA 6000 Nano chips.

treatment_protocol: Groups of one-year old male B6C57/J mice were maintained on ad libitum AIN-93G SD diet,(CONTROL) or a 40% caloric restriction of the AIN-93G SD diet,(CR) or an ad libitum AIN-93G diet supplemented with 0.1% w/w metformin,(METFORMIN) for the rest of their lives.

value: male

tag: strain

tag: diet

tag: tissue

value: Liver


title: 120629_PBMC_2


title: Liver_Ad_Lib_Metformin_Replicate-1

description:
Total RNA including miRNA
Liver_Ad_Lib_Metformin-1

tag: gender

value: ad libitum AIN-93G SD diet plus 0.1% w/w metformin


title: Liver_Ad_Lib_Metformin_Replicate-3

description:
Total RNA including miRNA
Liver_Ad_Lib_Metformin-3


title: Liver_Ad_Lib_Metformin_Replicate-4

description:
Total RNA including miRNA
Liver_Ad_Lib_Metformin-4


title: 120618_PBMC_2


title: Liver_Caloric_Restriction_Replicate-2

description:
Total RNA including miRNA
Liver_Caloric_Restriction-2

value: 40% caloric restriction of the AIN-93G SD diet


title: 120629_PBMC_0


title: Liver_Caloric_Restriction_Replicate-3

description:
Total RNA including miRNA
Liver_Caloric_Restriction-3


title: Liver_Ad_Lib_Control_Replicate-4

description:
Total RNA including miRNA
Liver_Ad_Lib_Control-4

value: ad libitum AIN-93G SD diet


title: 120618_PBMC_28


title: Liver_Caloric_Restriction_Replicate-4

description:
Total RNA including miRNA
Liver_Caloric_Restriction-4


title: Liver_Ad_Lib_Control_Replicate-5

description:
Total RNA including miRNA
Liver_Ad_Lib_Control-5


title: Liver_Ad_Lib_Metformin_Replicate-5

description:
Total RNA including miRNA
Liver_Ad_Lib_Metformin-5


title: Liver_Caloric_Restriction_Replicate-1

description:
Total RNA including miRNA
Liver_Caloric_Restriction-1


title: Liver_Ad_Lib_Control_Replicate-2

description:
Total RNA including miRNA
Liver_Ad_Lib_Control-2


title: Liver_Ad_Lib_Control_Replicate-1

description:
Total RNA including miRNA
Liver_Ad_Lib_Control-1


title: 120618_PBMC_7


title: Liver_Ad_Lib_Metformin_Replicate-2

description:
Total RNA including miRNA
Liver_Ad_Lib_Metformin-2


title: 120618_PBMC_0


title: 120660_PBMC_0


title: Liver_Ad_Lib_Control_Replicate-3

description:
Total RNA including miRNA
Liver_Ad_Lib_Control-3


title: S01_PDL54

status: Public on Feb 13 2017

description: Circular RNA

data_processing:
The reags were cleaned usning cutadapt program
The tags were aligned using Tophat and Tophat fusion (v2.1.0) with --GTF option and segment length of 20 (all other parameters were at their default values)
CIRCexplorer (v1.1.7) was used to find circRNAs using hg19 Ensembl v82 annotation database
Genome_build: hg19
Supplementary_files_format_and_content: The processed data file contain information about the circRNA and the junctional read counts in a tab-delimited TXT format.

source_name: human diploid WI-38 fibroblasts

growth_protocol: Human lung (WI-38) fibroblasts were obtained from Coriell Cell Repositories and cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), antibiotics, and non-essential amino acids (Invitrogen).  Proliferating WI-38 cells were used at population doubling (PDL) 15-25, and senescent fibroblasts were used after additional culture, at PDL 45-55.

extract_protocol:
RNA was isolated from WI-38 cells or from pulldown samples using TRIzol (Thermo Fisher Scientific) following the manufacturer’s procedure.
After isolation of total RNA using Trizol (Invitrogen) and assessment of quality and quantity using the Agilent 2100-Bioanalyzer, ribosomal RNA was removed by rRNA Depletion Nano kit (Qiagen) and cDNA was prepared and amplified using the Ovation RNA-Seq System V2 (NuGEN) kit following the manufacturer’s instructions.  The amplified cDNA was fragmented using a Bioruptor (Diagenode), adaptors were ligated to cDNA using TruSeq ChiP Sample Preparation kit (Illumina, San Diego, CA), the DNA fragments were size-selected (300-350 bp) after electrophoresis on a 4.5% agarose gel, and the selected DNA was subjected to 17 cycles of PCR amplification.  Library quality was determined on the Bioanalyzer 2100 and the final libraries were sequenced using the Illumina HiSeq2500 instrument.

treatment_protocol: No treatment

tag: tissue type

value: lung

value: senescent (late-passage)

value: WI-38 fibroblasts

tag: passages


title: S01_PDL50

description: Circular RNA


title: S01_PDL23

description: Circular RNA

value: proliferating (early-passage)


title: S01_PDL20

description: Circular RNA


title: cerebral_cortex_RESV_replicate 10

description:
RESV_WEJ
Streptavidin-Cy3 bound to biotin-labeled cRNA.

hyb_protocol: Standard Illumina protocol. In short, a total of 0.75ug of biotin-labeled cRNA was hybridized at 58 degrees C for 16 hours to Illumina's HumanHT-12 V4.0 expression BeadChip (Illumina, San Diego, CA). The arrays were washed, blocked and the biotin-labeled cRNA was detected by staining with streptavidin-Cy3.

scan_protocol: Arrays were scanned at a resolution of 0.8um using the BeadStation 500 X from Illumina.

data_processing: Data was extracted using the Illumina GenomeStudio software V2011.1, Gene expression module 1.9.0. Any spots at or below the background were filtered out using an Illumina detection Pvalue of 0.02 and above. The natural log of all remaining scores were used to find the avg and std of each array and the z-score normalization was calculated and presented below. Z-score = (raw value - avg)/std. Complete data including detection scores is supplied.

label: Biotin

source_name: cerebral cortex, HFS + Resveratrol (HFS+R)

label_protocol: Standard Illumina protocol using Illumina TotalPrep RNA Amplification Kit (Ambion; Austin, TX, cat # IL1791). In short, 0.5 ug of total RNA was first converted into single-stranded cDNA with reverse transcriptase using an oligo-dT primer containing the T7 RNA polymerase promoter site and then copied to produce double-stranded cDNA molecules. The double-stranded cDNA was cleaned and concentrated with the supplied columns and used in an overnight in-vitro transcription reaction where single-stranded RNA (cRNA) was generated and labeled by incorporation of biotin-16-UTP.

growth_protocol: Twenty-four adult (7-13 years old) male rhesus monkeys (Macaca mulatta) were housed individually in standard nonhuman primate caging, in a temperature-regulated room (25 ±1°C), humidity at 60 ± 20%, on a 12h light/12h dark cycle (lights on from 7:00 to 19:00) with ad libitum access to drinking water at the NIH Animal Center. All monkeys had extensive visual, auditory, and olfactory but limited tactile contact with monkeys housed in the same room. Monkeys were monitored minimally 3 times daily by trained animal care staff. All procedures and animal care were conducted in accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals in an AALAC-accredited facility. All experimental procedures were approved by the Animal Care and Use Committee of the NIA Intramural Program.

extract_protocol: Total RNA from rhesus monkey's temporal neocortex at 24-months of dietary intervention was extracted using Trizol reagent (Invitrogen) and further purified using RNeasy mini columns (Qiagen) using the standard manufacturer's protocols. Samples were assessed for quality and quantity using an Agilent 2100 Bioanlyzer with RNA 6000 Nano Chips (Agilent Technologies).

treatment_protocol: During baseline assessment, all monkeys were maintained on standard NIH monkey chow (Purina Mills). After baseline assessment, monkeys were quasi-randomized to one of three dietary regimens such that average age and bodyweight did not differ significantly between groups: control diet (C) group fed a healthy standard diet (SD) with 13% Kcal in fat and less than 5% sucrose by weight, high fat and sugar diet (HFS) group fed with 42% Kcal in fat and 27% sucrose by weight (Harlan, Teklad), and HFS + RSV (RESV) group fed with the same HFS diet plus a flavored primate treat (Bioserve) containing 40 mg and 240 mg resveratrol (RSV) twice daily, during year 1 and 2, respectively. Monkeys in non-RSV groups received placebo treats. All groups received 2 meals per day in allotments that represent ad libitum feeding. The HFS diet was formulated to cause weight gain and symptoms of metabolic syndrome, such as increased abdominal circumference, higher serum insulin levels after an intravenous glucose challenge, higher LDL-cholesterol but lower HDL-cholesterol levels compared to baseline levels, as well as lower insulin sensitivity after 24 months on a HFS diet. RSV was supplied by DSM Nutritional Products, Parsippany, NJ. At the end of the two-year dietary treatment period, monkeys were anesthesized with ketamine (7-10 mg/kg, IM). Following collection of blood and cerebrospinal fluid (CSF), animals were euthanized by an overdose of pentobarbital (50 mg/kg) and perfused with cold lactated Ringer's solution. Tissues were harvested immediately and frozen at -80°C. All procedures and animal care were conducted in accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals in an AALAC-accredited facility. All experimental procedures were approved by the Animal Care and Use Committee of the NIA Intramural Program.

tag: age

value: adult

value: brain (cerebral cortex)

value: high-fat/high-sugar

tag: treatment

value: resveratrol


title: cerebral_cortex_RESV_replicate 8

description:
RESV_TEJ
Streptavidin-Cy3 bound to biotin-labeled cRNA.


title: cerebral_cortex_SD_replicate 3

description:
SD_PXT
Streptavidin-Cy3 bound to biotin-labeled cRNA.

source_name: cerebral cortex, standard diet (SD)

value: standard

value: placebo


title: cerebral_cortex_SD_replicate 4

description:
SD_V14
Streptavidin-Cy3 bound to biotin-labeled cRNA.


title: cerebral_cortex_RESV_replicate 9

description:
RESV_TGF
Streptavidin-Cy3 bound to biotin-labeled cRNA.


title: cerebral_cortex_SD_replicate 2

description:
SD_P53
Streptavidin-Cy3 bound to biotin-labeled cRNA.


title: cerebral_cortex_SD_replicate 1

description:
SD_95E142
Streptavidin-Cy3 bound to biotin-labeled cRNA.


title: cerebral_cortex_RESV_replicate 7

description:
RESV_RQ4037
Streptavidin-Cy3 bound to biotin-labeled cRNA.


title: cerebral_cortex_RESV_replicate 6

description:
RESV_P57
Streptavidin-Cy3 bound to biotin-labeled cRNA.


title: cerebral_cortex_RESV_replicate 2

description:
RESV_96C029
Streptavidin-Cy3 bound to biotin-labeled cRNA.


title: cerebral_cortex_RESV_replicate 5

description:
RESV_P48
Streptavidin-Cy3 bound to biotin-labeled cRNA.


title: cerebral_cortex_RESV_replicate 4

description:
RESV_AV7W
Streptavidin-Cy3 bound to biotin-labeled cRNA.


title: cerebral_cortex_HFS_replicate 10

description:
HFS_TTJ
Streptavidin-Cy3 bound to biotin-labeled cRNA.

source_name: cerebral cortex, high-fat/high-sugar (HFS)


title: cerebral_cortex_HFS_replicate 8

description:
HFS_T83
Streptavidin-Cy3 bound to biotin-labeled cRNA.


title: cerebral_cortex_HFS_replicate 6

description:
HFS_RQ1784
Streptavidin-Cy3 bound to biotin-labeled cRNA.


title: worm_agestate3_late_replicate1

status: Public on Dec 19 2016

data_processing: Raw microarray hybridization intensity data from four separate experiments were log-transformed. Three separate probe preparations were used individually or combined in equal amounts for the fourth hybridization. Raw microarray data were log transformed to yield z-scores. The z-ratio was calculated as the difference between the observed gene z-scores for the experimental and the control comparisons, and dividing by the standard deviation associated with the distribution of these differences. Z-ratio values of ± 1.5 were used as cut-off values and calculated using a 5% false discovery rate (FDR) threshold. The complete set was tested for gene set enrichment using parametric analysis of gene set enrichment. For each pathway z-score, a p-value was computed using JMP 6.0 software to test for the statistical significance.

growth_protocol: Caenorhabditis elegans (C. elegans) nematodes were cultured on 'stock' plates, with 3 young adult worms transferred to a 6cm NGM agar plate spotted with OP50 strain of E. coli as the food source, and allowed to grow for 7 days at 15°C. Plates then contained a layer of 300-500 eggs on the remaining food, as well as individuals representing all stages of development. Five adults collected from the 'stock' plates were allowed to lay eggs on a fresh NGM plate at non-permissive temperature, 25°C, for the production of F2 progeny, Adults were removed after four hours. Eggs were counted and plates containing more than 100 eggs were noted. After 48 hours, L4 animals were transferred to fresh plates to avoid depletion of the bacterial lawn. Stock plates were washed three times with 1200ul M9 buffer and placed at 25°C for 4 hours. Washes were examined and discarded. If any eggs were present in the third wash, the plate was not used further. L1 hatchlings were collected by an additional 1200ul wash and brief centrifugation, 800 rcf for one minute. L1 hatchlings were carefully resuspended in 200 ul of fresh M9 and 40-80 individuals were plated on NGM agar seeded with OP50. These individuals were considered 12 hours older than the eggs derived from 'timed egglay', above. Synchronized animals were obtained from the same plate(s), anesthetized and imaged. A real-time classifier scored each animal’s age-state, guiding the sorting into collection tubes appropriate to the transition day being scored, and scores checked for each worm before they were picked from the agarose pad into chilled M9 buffer, and the process was repeated until at least 100 worms were sorted during each run. Collection tubes were centrifuged at 800 rcf for 1 minute, and all buffer removed under a dissecting microscope to avoid sample loss. Animals were frozen as a ‘dry’ pellet at -80C for storage until RNA preparation.

extract_protocol: Worm pellets were resuspended in 250 ul buffer RLT (Qiagen RNeasy MiniKit) and transferred to BeadBug tubes (2.0 ml  capacity with 0.5mm  Zirconium beads, triple-pure, Sigma-Aldrich) to the Precellys homogenizer using 3 cycles of 6000 rpm x 30 seconds, with two minutes rest between each cycle. Supernatant was collected, beads were washed with 150ul of fresh RLT and the consolidated supernatants were processed via Qiagen RNeasy columns. Total RNA was eluted into 40 ul water, and the quality and quantity of RNA was assessed by Nanodrop (ThermoFisher, Waltham, MA USA) and the Agilent Bioanalyzer RNA 6000 Chip (Agilent, Santa Clara, CA).

tag: included in analysis ?

value: Yes

tag: state

value: State  3 Late

value: Day 12


title: worm_agestate3_early_replicate5

value: State  3 Early

tag: day

value: Day 8


title: cerebral_cortex_HFS_replicate 2

description:
HFS_95E081
Streptavidin-Cy3 bound to biotin-labeled cRNA.

value: male


title: cerebral_cortex_RESV_replicate 1

description:
RESV_93N080
Streptavidin-Cy3 bound to biotin-labeled cRNA.


title: cerebral_cortex_HFS_replicate 7

description:
HFS_RQ1956
Streptavidin-Cy3 bound to biotin-labeled cRNA.


title: cerebral_cortex_RESV_replicate 3

description:
RESV_96C228
Streptavidin-Cy3 bound to biotin-labeled cRNA.


title: 120637_PBMC_7

value: OLD

value: R


title: cerebral_cortex_HFS_replicate 9

description:
HFS_TOK
Streptavidin-Cy3 bound to biotin-labeled cRNA.


title: worm_agestate3_late_replicate2


title: worm_agestate3_early_replicate2


title: worm_agestate3_late_replicate3


title: worm_agestate3_early_replicate4


title: worm_agestate3_early_replicate6

value: NO


title: cerebral_cortex_HFS_replicate 5

description:
HFS_R40
Streptavidin-Cy3 bound to biotin-labeled cRNA.


title: worm_agestate2_late_replicate2

value: State 2 Late


title: cerebral_cortex_HFS_replicate 1

description:
HFS_383
Streptavidin-Cy3 bound to biotin-labeled cRNA.


title: cerebral_cortex_HFS_replicate 3

description:
HFS_96C219
Streptavidin-Cy3 bound to biotin-labeled cRNA.


title: worm_agestate2_late_replicate4


title: cerebral_cortex_HFS_replicate 4

description:
HFS_M83
Streptavidin-Cy3 bound to biotin-labeled cRNA.


title: worm_agestate2_late_replicate3


title: worm_agestate2_early_replicate1

value: State 2 Early

value: Day 3


title: worm_agestate2_early_replicate3


title: worm_agestate2_early_replicate2


title: worm_agestate2_early_replicate4


title: worm_agestate3_early_replicate1


title: worm_agestate3_early_replicate3


title: worm_agestate2_late_replicate1


title: worm_agestate1_late_replicate4

value: State 1 Late


title: worm_agestate1_late_replicate2


title: worm_agestate1_late_replicate3


title: worm_agestate1_early_replicate3

value: State 1 Early

value: Day 1


title: HUVEC_Scrambled control_3

status: Public on Nov 16 2016

description: Sample name: Scrambled_3

hyb_protocol: Standard Illumina protocol. In short, a total of 0.75 ug of biotin-labeled cRNA was hybridized at 58°C for 16 hours to Illumina's Sentrix Human-HT12 v4 Expression Bead Chips (Illumina, San Diego, CA). Each BeadChip has more than 47,000 probes derived from the National Center for Biotechnology Information Reference Sequence (NCBI) RefSeq Release 38 (November 7, 2009) and other sources. The arrays were washed, blocked and the biotin labeled cRNA was detected by staining with streptavidin-Cy3.

scan_protocol: Arrays were scanned at a resolution of 0.53 um using the Illumina iScan scanner.

data_processing: Data was extracted using the Illumina GenomeStudio software V2011.1, Gene expression module 1.9.0. Any spots at or below the background were filtered out using an Illumina detection Pvalue of 0.02 and above. The natural log of all remaining scores were used to find the avg and std of each array and the z-score normalization was calculated and presented below.  Z-score = (raw value - avg)/std.  Complete data including detection scores is supplied.

label_protocol: Standard Illumina protocol using Illumina TotalPrep RNA Amplification Kit (Ambion; Austin, TX, cat # IL1791) In short, 0.5ug of total RNA was first converted into single-stranded cDNA with reverse transcriptase using an oligo-dT primer containing the T7 RNA polymerase promoter site and then copied to produce double-stranded cDNA molecules. The double stranded cDNA was cleaned and concentrated with the supplied columns and used in an overnight in-vitro transcription reaction where single-stranded RNA (cRNA) was generated and labeled by incorporation of biotin-16-UTP.

extract_protocol:
Primary human umbilical vein endothelial cells (HUVECs) were purchased from Lonza (Walkersville, MD) and grown in  EBM supplemented with the EGM SingleQuot Kit (Lonza).
Total RNA from HUVECs was isolated using TRIzol (Life Technologies) according to the manufacturer’s protocol. RNA quality was measured by a Nanodrop 2000 and Agilent Bioanalyzer and all RNA was stored at -80°C.

treatment_protocol: HUVECs were transfected with 50 nM Pre-miR miRNA Precursors for hsa-miR-4717-3p, -4709-3p, or scrambled negative control (Life Technologies) using Lipofectamine 2000 (Life Technologies).

tag: transfection

value: scrambled negative control


title: HUVEC_hsa_miR4717_3p_replicate_2

description: Sample name: 4717_2

source_name: Primary human umbilical vein endothelial cells

value: pre-miR4717 miRNA precursor


title: HUVEC_Scrambled control_2

description: Sample name: Scrambled_2


title: worm_agestate1_late_replicate1

value: Yes


title: HUVEC_Scrambled control_1

description: Sample name: Scrambled_1


title: worm_agestate1_early_replicate1


title: HUVEC_hsa_miR4709_3p_replicate_3

description: Sample name: 4709_3

value: pre-miR4709 miRNA precursor


title: HUVEC_hsa_miR4709_3p_replicate_1

description: Sample name: 4709_1


title: HUVEC_hsa_miR4717_3p_replicate_1

description: Sample name: 4717_1


title: HUVEC_hsa_miR4717_3p_replicate_3

description: Sample name: 4717_3


title: worm_agestate1_early_replicate2


title: HUVEC_hsa_miR4709_3p_replicate_2

description: Sample name: 4709_2


title: HUVEC_hsa_miR4763_replicate_3

description: Sample name: miR-4763_3

treatment_protocol: HUVECs were transfected with 50 nM Pre-miR miRNA Precursors for hsa-miR-4763-5p, -30c-5p, -20a-5p or scrambled negative control (Life Technologies) using Lipofectamine 2000 (Life Technologies).

value: pre-miR4763 miRNA precursor


title: HUVEC_hsa_miR30c_replicate_2

description: Sample name: miR-30c_2

value: pre-miR30c miRNA precursor


title: PBMC_normotensive_CAU_case_2

description: Sample name: WNHT-1627

source_name: white normotensives

extract_protocol:
Fasting blood samples were obtained from participants in the Healthy Aging in Neighborhoods of Diversity across the Life Span (HANDLS) study of the National Institute on Aging Intramural Research Program (NIA IRP), National Institutes of Health (NIH). This study was approved by the Institutional Review Board of the National Institute of Environmental Health Sciences (NIEHS), NIH and all participants provided written informed consent. Age-matched AA and white females that were either normotensive (NT) or hypertensive (HT). Hypertension was defined as an average SBP ≥140 mmHg or DP ≥90 mmHg or use of previously prescribed antihypertensive medications (diuretics, beta blockers, ACE inhibitors, angiotensin II blockers, calcium channel blockers, or vasodilators) and/or prior diagnosis of hypertension. We excluded participants with documented Hepatitis B, Hepatitis C, or human immunodeficiency virus (HIV) infection and current or former smokers.
Total RNA from PMBCs was isolated using the miRVana miRNA Isolation Kit with enrichment for small RNAs according to the manufacturer’s protocol (Life Technologies). RNA quality was measured by a Nanodrop 2000 and Agilent Bioanalyzer and all RNA was stored at -80°C.

value: Female

tag: hypertension status

value: Normotensive

tag: race

value: Caucasian

value: PBMC


title: PBMC_hypertensive_CAU_case_5

description: Sample name: WHT-924

source_name: white hypertensives

value: Hypertensive


title: HUVEC_hsa_miR30c_replicate_3

description: Sample name: miR-30c_3


title: HUVEC_hsa_miR20a_replicate_1

description: Sample name: miR-20a_1

value: pre-miR20a miRNA precursor


title: HUVEC_hsa_miR20a_replicate_3

description: Sample name: miR-20a_3


title: HUVEC_Control_Replicate_2

description: Sample name: Control_3


title: HUVEC_Control_Replicate_1

description: Sample name: Control_1


title: HUVEC_hsa_miR20a_replicate_2

description: Sample name: miR-20a_2


title: PBMC_normotensive_CAU_case_4

description: Sample name: WNHT-523


title: PBMC_normotensive_CAU_case_5

description: Sample name: WNHT-949


title: PBMC_normotensive_CAU_case_3

description: Sample name: WNHT-2172


title: PBMC_hypertensive_CAU_case_3

description: Sample name: WHT-2278


title: PBMC_hypertensive_CAU_case_4

description: Sample name: WHT-2330


title: HUVEC_hsa_miR4763_replicate_2

description: Sample name: miR-4763_2


title: PBMC_normotensive_CAU_case_1

description: Sample name: WNHT-1185


title: PBMC_normotensive_AA_case_1

description: Sample name: BNHT-1049

source_name: African American_normotensives

value: African American


title: PBMC_normotensive_AA_case_5

description: Sample name: BNHT-808


title: PBMC_hypertensive_CAU_case_1

description: Sample name: WHT-1112


title: PBMC_hypertensive_AA_case_5

description: Sample name: BHT-953

source_name: African American_hypertensives


title: PBMC_normotensive_AA_case_3

description: Sample name: BNHT-760


title: PBMC_normotensive_AA_case_2

description: Sample name: BNHT-381


title: PBMC_hypertensive_AA_case_4

description: Sample name: BHT-1759


title: PBMC_hypertensive_AA_case_2

description: Sample name: BHT-1515


title: PBMC_hypertensive_AA_case_3

description: Sample name: BHT-1577


title: PBMC_normotensive_AA_case_4

description: Sample name: BNHT-775


title: PBMC_hypertensive_CAU_case_2

description: Sample name: WHT-1378


title: PBMC_normotensive_AA_case_6

description: Sample name: BNHT-843


title: PBMC_hypertensive_AA_case_1

description: Sample name: BHT-1389


title: Senescent mitoplast

status: Public on Sep 12 2016

description: mitoplast RNA

data_processing:
The tags were aligned using Tophat (v2.0.14) with --GTF option and segment length of 20 (all other parameters were at their default values)
The aligned tags were assebled using Cufflinks (v2.2.1) with --GTF-guide option and minimum fragment treshold of 5 (all other parameters were at their default values)
The samples were merged using Cuffcompare
Significant changes were calculated using Cuffdiff using fdr cuttoff of 0.1
Genome_build: hg19
Supplementary_files_format_and_content: The processed data file contain the FPKM values of the isoforms in tab-delimited TXT format.

source_name: WI-38 fibrobalst (PDL 60)

growth_protocol: WI-38 human fibloblasts were maintained in DMEM supplemented with 10% FBS, 1% Anti-anti, and 1% NEAA at 37oC in CO2 incubator

extract_protocol:
RNA was isolated from whole or mitoplast fraction of cultured young and senescent WI-38 cells. To obtain mitoplast fraction, crude mitochondria were first isolated from cultured WI-38 cells.  Briefly, cells were scraped, centrifuged, and washed once with ice-cold PBS.  After subsequent centrifugation, the cell pellets were resuspended in a hypotonic buffer (20 mM HEPES [pH 7.3], 1.5 mM MgCl2, 10 mM KCl), vortexed at low speed for 5 seconds, and incubated on ice for 2 min.  CHAPS buffer (20 mM HEPES [pH 7.3], 8% (w/v) CHAPS) was added and vortexed vigorously 5 times while incubating on ice for 5 min.  Hypertonic buffer (20 mM HEPES [pH 7.3], 420 mM mannitol, 140 mM sucrose, 2 mM EGTA) was then added with equal volume of hypotonic buffer, and gently mixed by inverting the tubes several times.  After centrifugation at 2,000 x g, supernatants were collected and centrifuged again at 2,000 x g for 10 min to remove nuclei.  From the supernatant, mitochondria were pelleted down at 12,000 x g for 15 min.  The crude mitochondria were then incubated with hypotonic buffer on ice for 30 min, whereupon the same volume of hypertonic buffer was added to stop the swelling.  After the centrifugation, samples were treated with RNase A (2 mg/mL) (Life technologies) to remove nuclear and cytosolic RNAs, as previously described (Mercer et al., 2011).  RNase A activity was neutralized by addition of proteinase K (PK; 50 μg/mL).  RNaseOUT (Invitrogen) was used to completely inhibit RNase A activity before RNA isolation.  Mitoplast pellet was obtained by centrifugation at 12,000 x g and treated with RNase A again as described above.  Purified mitoplast pellets were incubted in mitochondria solubilizing buffer at 65 ºC for 5 min.  RNA was isolated using TRIzol reagent, and treated with RNase-free DNase I (Roche) for 1 h at 37 ºC.  RNA was then extracted by acidic phenol (Ambion).
RNASeq library was prepared by using TruSeq™ RNA Sample Preparation V2 Kit (Illumina, Cat #: RS-122-2001, RS-122-2002). Poly-A RNA was first purified from 100 ng high quality total RNAs, fragmented to about 150-200 nucleotides in length; then go for 1st-strand and 2nd-strand cDNA synthesis, end repair to remove remove 3’ end overhangs and fill in 5’ overhangs; next an “A” base is added to the 3’ end for adaptor ligation and PCR enrichment (libraries); finally quantify library and check the library quality on Bioanalyzer using DNA 1000 chips. High sensitivity chips are used for further quality control and ensure adequate concentration and optimal fragment size.

tag: cellular fraction

value: Mitoplast

tag: cell line


title: Young mitoplast

description: mitoplast RNA

source_name: WI-38 fibrobalst (PDL 21)


title: Senescent whole cells

description: total RNA

value: Whole cells


title: Young whole cells

description: total RNA


title: AA_hypertensive_pool_replicate_1

description: Sample name: BHT

hyb_protocol: Exiqon miCURY 7th generation LNA microRNA arrays were hybridized for 16 hours at 56oC using the Agilent SureHyb chamber and gasket slide kit, as per the manufacturer's protocol.

scan_protocol: Arrays were scanned on an Agilent scanner at 10 micron resolution.

data_processing: Spot evaluation and data extraction was done using Agilent Feature Extraction software without background subtraction as per the manufacturer's protocol. Any spots at or below the background were filtered out. The natural log of all remaining scores were used to find the avg and std of each array and the z-score normalization was calculated and presented below.  Z-score = (raw value - avg)/std.

source_name: Blood

label_protocol: One microgram total RNA containing small RNA species was pooled from three-four individuals, such that forty individuals were sampled via three replicate pools for each of the four disease state x ethnicity comparisons. This pooled RNA was labeled according to the manufacturer's protocol, directly incorporating the Hy3-flourescent label

growth_protocol: Fasting blood samples were obtained from participants in the Healthy Aging in Neighborhoods of Diversity across the Life Span (HANDLS) study of the National Institute on Aging Intramural Research Program (NIA IRP), National Institutes of Health (NIH). This study was approved by the Institutional Review Board of the National Institute of Environmental Health Sciences (NIEHS), NIH and all participants provided written informed consent. Age-matched AA and white females that were either normotensive (NT) or hypertensive (HT). Hypertension was defined as an average SBP ≥140 mmHg or DP ≥90 mmHg or use of previously prescribed antihypertensive medications (diuretics, beta blockers, ACE inhibitors, angiotensin II blockers, calcium channel blockers, or vasodilators) and/or prior diagnosis of hypertension. We excluded participants with documented Hepatitis B, Hepatitis C, or human immunodeficiency virus (HIV) infection and current or former smokers.

extract_protocol: Total RNA from PMBCs was isolated using the miRVana miRNA Isolation Kit with enrichment for small RNAs according to the manufacturer’s protocol (Life Technologies). RNA quality was measured by a Nanodrop 2000 and Agilent Bioanalyzer and all RNA was stored at -80°C.

tag: ethnicity


title: White_hypertensive_pool_replicate_2

description: Sample name: WHT 2