title: EP1-S258-CD56 vs. EP1-HREF

hyb_protocol: [Tecan HS4800 hybridization]  * 60 ul labeled sample is combined with 60 ul 2x-hybmix, containing 50% formamide, 10xSSC, 0.2% SDS, 200 ug/ml herring sperm DNA    * Hybridizations of spotted oligo-arrays (Codelink glass) or Agilent microarrays are performed on a HS4800Pro Hybstation (Tecan)    * Priming: 5xSSC, 0.1%SDS    * Probe injection: pre-hyb, 5xSSC, 25% formamide, 0.1%SDS, 1%BSA, Volume 110ul    * Hybridization: 45 min at 42C.    * Wash 2x: milliQ    * Wash: 5xSSC, 0.1%SDS    * Probe injection: sample. Volume 110ul (Agilent 4packs: 55ul)    * Hybridization: 16 hours at 42C.    * Wash 2x: 1xSSC, 0.2%SDS at 23C    * Wash 2x: 0.1xSSC, 0.2%SDS at 23C    * Wash 2x: 0.1xSSC at 23C    * Drying: blow with nitrogen for 3min at 30C

scan_protocol: Scanning of slides using the Agilent G2565BA scanner. [Imagene feature extraction] Features were extracted using Imagene software from Biodiscovery.

data_processing: [genes, no bg corr] Normalization was done using print-tip LOESS as described in (Yang et al. 2002), for all gene probes using no background substraction and a span of 0.4. Probes flagged as absent, or with a (nearly) saturated signal (i.e., > 2^15) in either channel were not considered for the estimation of the LOESS curve.

label_protocol: [robot amplification and labeling v1.0]  All amplification and labeling procedures are performed in 96 wells plates (4titude, Bioke) on a customized Sciclone ALH 3000 Workstation (Caliper LifeSciences), supplemented with a PCR PTC-200 (Bio-Rad Laboratories), SpectraMax 190 spectrofotometer (Molecular Devices), and a magnetic bead-locator (Beckman).        mRNA amplification protocol description:    * all RNAs are diluted to 0.6 ug/ul and 5 ul is put in a 96-wells plate (Abgene). All subsequent steps are performed by the robot script.    * mix1 containing 100 ng T7 Mlu VN primer (custom mix) and EC-control RNA per 5 ul is added and mixed in each well.    * plate is incubated @ 70C for 10 mins, cooled to 48C.    * mix2 containing 4ul 5x 1st strand buffer, 2 ul 0.1M DTT (Invitrogen), 1ul RNAse Inhibitor (Boehringer), 1ul 20mM dNTPs (GE Healthcare), 1ul linear acrylamide, and  1ul SuperScriptIII (Invitrogen) per sample is prewarmed to 48C; 10 ul per sample is added and mixed in each well.    * plate is incubated @ 48C for 2 hours and cooled to room temperature.    * 106 ul water and subsequently  mix3 containing 15ul second strand buffer, 3ul 20mM dNTPs (GE Healthcare), 1 ul T4 DNA ligase, 4 ul E.coli DNA polymerase I and 1 ul RNAseH  (Promega) is added and mixed in each well.    * plate is incubated @ 16C for 2 hours, @65C for 10 mins. * ds cDNA product is purified and concentrated with RNAClean (Agencourt, GC biotech) according to manufacturers protocol, to an end volume of 25 ul .    * 8 ul cDNA is put in a 96 wells plate and mixed with 12 ul IVT mix containing 2 ul 10x rxn-buffer, 2 ul each ATP, CTP, GTP, 0.6 ul UTP, 2 ul T7 enzyme mix (MegaScript kit, Ambion, Applied Biosystems), and 2.1 ul 50 mM 5-(3-aminoallyl)-UTP (Ambion, Applied Biosystems).    * Plate is incubated @37C for 4 hours.    * cRNA product is purified with RNAClean (Agencourt, Beckman) according to manufacturers protocol.     * Concentration  is measured (SpectraMax 190) and adjusted to 600 ng/ul by the robot.    * Resulting cRNA plates are sampled for Bioanalyzer QC, snapfrozen and stored at -80C.        Labeling protocol description:     * NHS-ester Cy3 or Cy5 dye (Amersham PA 23001 and 25001): entire tube is resuspended in 100 ul DMSO (Merck 8.02912.10).      All subsequent steps are performed by the robot script :    * 8 ul of each cRNA (0.6 ug/ul) is put in a 96-wells plate (Abgene), the accompanying reference sample is put in the next column (final step combines cy3 and cy5 labeled material ).    * 3 ul 0.5 M NaBicarbonate buffer, pH 9 is added and mixed to all wells.    * 3 ul cy-dye solution is added and mixed to the appropriate wells, plate is incubated at 18C for 1 hour.    * 4.5 ul 5M hydroxylamine is added and mixed, incubated at 18o C for 15 minutes.    * Labeled cRNA product is purified with RNA Clean (Agencourt, GC biotech) according to manufacturers protocol.    * RNA concentration and labeling incorporation are measured (SpectraMax 190).    * 2.5 ug of each labeled sample and reference cRNA are pooled and subjected to fragmentation according to protocol (Ambion, Applied Biosystems), 15 min 70C.    * Samples are stored at -20C until hybridization.

extract_protocol: [RNA extraction cellLine]  1. Add 1 ml Trizol per 5x 106 cells in suspension (1 ml trizol per 10 cm culture disk) and mix by pipetting, transfer to 1.5 ml eppendorf tube.     2. Incubate 5 min at room temperature.     3. Add chloroform (200 Âµl per ml Trizol), mix for 15 seconds by shaking vigorously. Incubate 2-3 min at room temperature.     4. Centrifuge 20 min at 14000 rpm 4Â°C. After centrifugation, transfer the water-phase to a new 1.5-ml micro centrifuge tube.     5. add isopropanol to precipitate RNA. Use 0.5 ml per 1 ml trizol. Mix by shaking, leave samples at RT for 10 minutes. Centrifuge at 14000 rpm for 15 min.     6. wash pellet with 75% ethanol. Centrifuge at 14000 rpm for 15 min. Carefully pipet off all ethanol (respin briefly and use a P20 for the last microliters). Airdry pellets briefly before dissolving in MilliQ water. Depending on pellet-size, this may take a while. Continue with RNeasy columns (Qiagen) for clean-up of RNA (maximal 100 Âµg/column)     7. Adjust each sample to a volume of 300 Âµl with sterile MilliQ water. Add 160 Âµl buffer RLT to the samples, and mix (= lysis buffer).     8. Add 240 Âµl ethanol (100% stored at -20 Â°C) to the lysate, mix well by pipetting and immediately apply the sample to an RNeasy mini spin column sitting in a 2 ml collection tube (use a second P1000 pipet and work as quick as possible). Next lysate, and so on.     9. Centrifuge at 8000 rpm for 15 seconds in an Eppendorf centrifuge. Discard flowthrough, reuse collection tube. All centrifugation steps at roomtemperature! DNAse treatment: D1. Pipet 350 Âµl Buffer RW1 into the RNeasy mini column, and centrifuge for 15 s at 8000 rpm to wash. Discard the flow-through. Reuse the collection tube in step D3. D2. Prepare DNase I incubation mix (80 Âµl/reaction): add 10 Âµl DNase I stock solution to 70 Âµl Buffer RDD. Mix by gently inverting the tube. Buffer RDD is supplied with the RNase-Free DNase Set. Note: DNase I is especially sensitive to physical denaturation. Mixing should only be carried out by gently inverting the tube. Do not vortex. D3. Pipet the DNase I incubation mix (80 Âµl) directly onto the RNeasy silica-gel membrane, and place on the benchtop (20-30Â°C) for 15 min. Note: Make sure to pipet the DNase I incubation mix directly onto the RNeasy silica-gel membrane. DNase digestion will be incomplete if part of the mix sticks to the walls or the O-ring of the RNeasy column. D4. Pipet 350 Âµl Buffer RW1 into the RNeasy mini column, and centrifuge for 15 s at 8000 rpm. Discard the flow-through and collection tube.     10. Transfer the RNeasy column into a new 2 ml collection tube. Add 500 Âµl buffer RPE, wait 1 minute, and centrifuge at 8000 rpm for 15 seconds. Discard flow-through and reuse the collection tube in next steps.    11. Pipet 500 Âµl buffer RPE onto the RNeasy column, wait 1 minute, and centrifuge at 8000 rpm for 15 seconds. Discard flow-through and reuse collection tube.     12. Centrifuge at 10,000 rpm for 2 minutes (to dry the RNeasy membrane).     13. Transfer the RNeasy column into a new 1.5 ml collection tube. Pipet 100 Âµl milliQ directly onto the RNeasy membrane and incubate for 4 minutes at room temperature. Centrifuge at 14,000 rpm for 1 minute to elute. Note: When small yields are expected, the elution volume can be lowered to a minimum of 35 Âµl milliQ.     14. Pipet the eluate of step 13 again onto the RNeasy membrane and incubate for another 4 minutes at room temperature. Centrifuge at 14,000 rpm for 1 minute to elute. After elution, put all RNA-samples on ice. 15. Determine RNA concentration by measuring A260, and RNA intactness by Bioanalyzer. Snap-freeze and store eluate at -80 Â°C.

treatment_protocol: [uNK sorting procedure]  From decidual tissue samples we purified CD56brightCD16-CD3- uNK cells with an enzymatic digestion protocol and positive selection with a BD FACSAriaTM III cell sorter (BD Biosciences, Franklin Lakes, NJ), with a gating strategy adapted from Hanna et al [Hanna 16892062]. To verify the resulting uNK cell purity, cells were analyzed immediately after cell sorting with the same fluorescence and compensation values used in their initial sorting.

value: CD56-positive uNK cells

tag: trimester

tag: sample type

value: Stratagene Universal Human Reference RNA


title: EP1-S257-CD56 vs. EP1-HREF

status: Public on Jan 30 2020

type: RNA

city: Utrecht

first: Marian

last: Groot Koerkamp

state: Utrecht

address: Heidelberglaan 25

department: Research

country: Netherlands

institute: Princess Maxima Center for Pediatric Oncology

molecule: total RNA

organism: Homo sapiens

tag: cell type


title: Adolescent Infarct 3 [LI3]

city: Manchester

first: Mitchell

middle: C

last: Lock

address: 46 Grafton Street

department: Division of Cardiovascular Sciences

country: United Kingdom

institute: University of Manchester

hyb_protocol: Fragmentation and hybridization were performed using protocols from Agilent. 600 ng of each labeled cRNA was fragmented and then mixed with hybridization buffer using the Agilent Gene Expression Hybridization Kit.

scan_protocol: The labelled cRNA was hybridized to Agilent 8 × 15k microarray slides (Ovine 019221 Arrays; GPL14112 platform, containing eight arrays with 15,744 oligomers each with a length of 60 bases and hybridized at 65°C for 17 h at 10 rpm), washed, and scanned with an Agilent G2505B 2 dye scanner at the Interdisciplinary Center for Biotechnology Research Core at the University of Florida.

data_processing: Raw microarray data were imported into R (http://www.r-project.org) using the limma package for background correction and normalization using the quantile normalization method. 7,631 probes on the microarray were excluded from further analysis due to low expression (probes were filtered out if they were not at least 10% stronger in signal than the negative controls on at least four arrays).

organism: Ovis aries

source_name: Adolescent sheep mycardial infarction three day infarct sample

label_protocol: 1 μg of DNase-treated RNA was labelled with cyanine 3 (Cy3) CTP using the Agilent Quick Amp kit (Agilent, USA) and purified with the Qiagen RNeasy kit (Qiagen, Germany), as previously described. The labelled cRNA was analyzed with the Nanodrop spectrophotometer, and the specific (Cy3) activities and the yields of the cRNAs were calculated and stored at -80°C until use.

extract_protocol: RNA was extracted from each ventricular tissue area. RNA concentration was determined using a Nanodrop spectrophotometer (ND-1000, ThermoFisher, Wilmington DE) and its integrity using an Agilent Bioanalyzer, 2100 model. RNA Integrity Number (RIN) values were between 7.6 and 9.5 for all samples.

tag: developmental stage

value: Adolescent

tag: tissue area

value: Infarct


title: NML1_Lin

type: SRA

city: San Francisco

first: Tatsuya

last: Tsukui

state: CA

address: 555 Mission Bay Blvd South, 282

department: Medicine

country: USA

institute: UCSF

library_source: transcriptomic

data_processing:
Sequencing data were demultiplexed, aligned to the mouse genome mm10 or human genome GRCh38, and UMI-collapsed with the Cell Ranger toolkit (version 2.0 or 3.0, 10X Genomics).
Genome_build: mm10 (mouse), GRCh38 (human)
Supplementary_files_format_and_content: barcodes.tsv: barcodes
Supplementary_files_format_and_content: genes.tsv: genes
Supplementary_files_format_and_content: matrix.mtx: gene-barcode matrix

source_name: Lung

extract_protocol:
Lung tissues were minced into 1 x 1 x 1 mm pieces and digested with protease solution (Collagenase A, Dispase II, DNase I in RPMI with 10 mM HEPES) at 37°C for 60 min with trituration every 20 min. Cells were stained with antibodies. For murine samples, Col-GFP+ or Col-GFP- cells were sorted. For human samples, lineage (CD31, CD45, CD235a, EPCAM)- cells, or all cells (CD235a-) were sorted. Approximately 10,000 sorted cells were loaded onto 10x Genomics Chromium controller.
Single cell libraries were generated using Chromium Single Cell 3' v2 Reagent Kit (10x Genomics) according to the manufacturer's protocol. The libraries were sequenced on Illumina HiSeq 4000 for murine samples or NovaSeq 6000 for human samples.

tag: disease diagnosis

value: Normal

tag: sorted cell type

value: Lineage-

tag: individual identifier


title: Fetal Sham 3 [FS3]

source_name: Fetal sheep sham surgery sample

value: Fetal

value: Sham


title: Fetal Remote 3 [FR3]

source_name: Fetal sheep mycardial infarction three day remote sample

value: Remote


title: Adolescent Border 3 [LB3]

source_name: Adolescent sheep mycardial infarction three day border sample

value: Border


title: NML2_Lin


title: Adolescent Remote 3 [LR3]

source_name: Adolescent sheep mycardial infarction three day remote sample


title: Fetal Border 3 [FB3]

source_name: Fetal sheep mycardial infarction three day border sample


title: Adolescent Sham 3 [LS3]

source_name: Adolescent sheep sham surgery sample


title: EP1-S256-CD56 vs. EP1-HREF


title: EP1-S255-CD56 vs. EP1-HREF


title: EP1-S253-CD56 vs. EP1-HREF


title: EP1-S254-CD56 vs. EP1-HREF


title: EP1-S251-CD56 vs. EP1-HREF


title: Fetal Infact 3 [FI3]

source_name: Fetal sheep mycardial infarction three day infarct sample


title: EP1-S249-CD56 vs. EP1-HREF


title: CHD9: 9 1ary Visual (BA17)

city: Moscow

first: Pavel

last: Mazin

address: Skolkovo Innovation Center, Building 3

department: Biomed

country: Russia

institute: Skolkovo Institute of Science and Technology

data_processing:
Read trimming: trimmomatic-0.36 PE -phred33 ILLUMINACLIP:all.fa:2:30:10:2:true SLIDINGWINDOW:4:15 LEADING:3 TRAILING:3 MINLEN:20
Read alignment: hisat2 --no-softclip --max-intronlen 1000000 -k 20 –no-unal -q
abundance (TPM) estimation: stringtie -e -A -G -b; TPM outputed by -A option was used as expression estimates and provided in processed data files
Genome_build: Ensemble genomes and annotation were used for all species: GRCh38.91, Pan_tro_3.0.91, panpan1.1.91, Mmul_8.0.1
Supplementary_files_format_and_content: csv files with TPM for all samples

molecule: polyA RNA

organism: Pan troglodytes

source_name: 9  1ary Visual (BA17)

extract_protocol:
Total RNA was isolated using Trizol reagent (Invitrogen, Carlsbad, California).
Sequencing libraries were prepared with TruSeq RNA Sample Preparation Kit (Illumina) according to manufacturer’s instruction. Briefly, poly-T oligo-attached magnetic beads were used to isolate long polyadenylated RNA from 1 μg of total RNA. After fragmentation, first-strand cDNA was reverse transcribed with random hexamer-primers, followed by second-strand cDNA synthesis, end repair, adenylation of 3′ ends, and ligation of the adapters. Fragments were then enriched by PCR.

tag: individual

value: cd

tag: brain region

value: 9  1ary Visual (BA17)


title: Adolescent Sham 2 [LS2]


title: CHD8: 4 Cingulate Posterior (BA31)

source_name: 4  Cingulate Posterior (BA31)

value: 4  Cingulate Posterior (BA31)


title: CHD5: 11 Cingulate Anterior (BA24)

source_name: 11 Cingulate Anterior (BA24)

value: 11 Cingulate Anterior (BA24)


title: Adolescent Remote 2 [LR2]


title: CHD41: 33 Nucleus Accumbens

source_name: 33 Nucleus Accumbens

value: 33 Nucleus Accumbens


title: CHD40: 27 Globus Pallidus

source_name: 27 Globus Pallidus

value: 27 Globus Pallidus


title: CHD42: 19 Amygdala

source_name: 19 Amygdala

value: 19 Amygdala


title: CHD4: 12 Cingulate Anterior (BA32)

source_name: 12 Cingulate Anterior (BA32)

value: 12 Cingulate Anterior (BA32)


title: Fetal Infact 2 [FI2]


title: Adolescent Border 2 [LB2]


title: Fetal Border 2 [FB2]


title: Adolescent Remote 1 [LR1]


title: Adolescent Infarct 2 [LI2]


title: CHD39: 26 Internal Capsule

source_name: 26 Internal Capsule

value: 26 Internal Capsule


title: CHD38: 31 Putamen

source_name: 31 Putamen

value: 31 Putamen


title: CHD37: 32 Caudate

source_name: 32 Caudate

value: 32 Caudate


title: Adolescent Border 1 [LB1]

value: Adolescent


title: Fetal Sham 2 [FS2]

source_name: Fetal sheep sham surgery sample


title: CHD7: 3 Precuneus (BA7)

source_name: 3  Precuneus (BA7)

value: 3  Precuneus (BA7)


title: BC42: 19 Amygdala

organism: Pan paniscus

value: bc


title: Fetal Remote 2 [FR2]

source_name: Fetal sheep mycardial infarction three day remote sample

value: Fetal


title: BC43: 29 Substantia Nigra

source_name: 29 Substantia Nigra

value: 29 Substantia Nigra


title: CHD6: 5 Premotor (BA6)

source_name: 5  Premotor (BA6)

value: 5  Premotor (BA6)


title: EP1-S211-CD56 vs. EP1-HREF

value: CD56-positive uNK cells

value: Stratagene Universal Human Reference RNA


title: EP1-S245-CD56 vs. EP1-HREF


title: EP1-S231-CD56 vs. EP1-HREF


title: EP1-S217-CD56 vs. EP1-HREF


title: BB42: 19 Amygdala

value: bb


title: CHD34: 21 Hippocampus

source_name: 21 Hippocampus

value: cd

value: 21 Hippocampus


title: BA42: 19 Amygdala

value: ba


title: CHD32: 15 Insular Posterior Cortex

source_name: 15 Insular Posterior Cortex

value: 15 Insular Posterior Cortex


title: MB43: 29 Substantia Nigra

organism: Macaca mulatta

value: mb


title: MC42: 19 Amygdala

value: mc

value: 19 Amygdala


title: MA43: 29 Substantia Nigra

value: ma


title: BA43: 29 Substantia Nigra


title: CHD31: 7 1ary Auditory (BA41/42)

source_name: 7  1ary Auditory (BA41/42)

value: 7  1ary Auditory (BA41/42)


title: BC41: 33 Nucleus Accumbens

value: 33 Nucleus Accumbens


title: CHD30: 6 1ary Somatosensory (BA3/1/2)

source_name: 6  1ary Somatosensory (BA3/1/2)

value: 6  1ary Somatosensory (BA3/1/2)


title: MA42: 19 Amygdala


title: MB42: 19 Amygdala


title: CHD3: 17 Orbitofrontal (BA11)

source_name: 17 Orbitofrontal (BA11)

value: 17 Orbitofrontal (BA11)


title: MC43: 29 Substantia Nigra

value: 29 Substantia Nigra


title: MC41: 33 Nucleus Accumbens

source_name: 33 Nucleus Accumbens


title: BB41: 33 Nucleus Accumbens


title: BA41: 33 Nucleus Accumbens


title: CHD29: 8 1ary Motor (BA4)

source_name: 8  1ary Motor (BA4)

value: 8  1ary Motor (BA4)


title: BC40: 27 Globus Pallidus

source_name: 27 Globus Pallidus

value: bc

value: 27 Globus Pallidus


title: CHD36: 20 Entorhinal Cortex

source_name: 20 Entorhinal Cortex

value: 20 Entorhinal Cortex


title: MB41: 33 Nucleus Accumbens


title: BB40: 27 Globus Pallidus

value: bb


title: donor50_Mock_day14

city: Padova

first: Silvio

last: Bicciato

address: via U. Bassi 59/b

department: Molecular Medicine

country: Italy

institute: University of Padova

data_processing:
Basecalls performed using CASAVA.
Reads were aligned using STAR version 2.5 to build version hg38 of the human genome and to calculate counts for GENCODE (release 25) basic annotated genes.
Normalization and differential analysis were carried out using the DESeq2 R package.
Genome_build: hg38
Supplementary_files_format_and_content: Matrix containing gene counts for each sample

source_name: Primary CD4 positive T-cell

growth_protocol: RPMI + 20% FCS + Il-2

extract_protocol:
InviTrap Spin Universal RNA Mini Kit
Ribozero, PE75, NextSeq 500

treatment_protocol: for infected samples: 2 ng/p24 per million cells

tag: donor

tag: treatment

value: Mock

tag: time point


title: BA40: 27 Globus Pallidus

value: ba


title: CHD28: 1 Supramarginal (BA40)

source_name: 1  Supramarginal (BA40)

value: cd

value: 1  Supramarginal (BA40)


title: MA41: 33 Nucleus Accumbens


title: CHD22: 30 Cerebellar Grey Matter

source_name: 30 Cerebellar Grey Matter

value: 30 Cerebellar Grey Matter


title: donor50_Mock_day9


title: MC40: 27 Globus Pallidus

value: mc


title: donor50_Mock_day7


title: BC39: 26 Internal Capsule

source_name: 26 Internal Capsule

value: 26 Internal Capsule


title: MC39: 26 Internal Capsule


title: BB39: 26 Internal Capsule


title: CHD20: 28 Cerebellar White Matter

source_name: 28 Cerebellar White Matter

value: 28 Cerebellar White Matter


title: CHD2: 16 Dorsolateral Prefrontal (BA9)

source_name: 16 Dorsolateral Prefrontal (BA9)

value: 16 Dorsolateral Prefrontal (BA9)


title: MB39: 26 Internal Capsule

value: mb


title: MA39: 26 Internal Capsule

value: ma


title: donor50_Infected_day14

value: Infected


title: CHD18: 22 Hypothalamus

source_name: 22 Hypothalamus

value: 22 Hypothalamus


title: CHD17: 23 Thalamus

source_name: 23 Thalamus

value: 23 Thalamus


title: BC38: 31 Putamen

source_name: 31 Putamen

value: bc

value: 31 Putamen


title: donor50_Infected_day9


title: donor50_Infected_day7


title: MC38: 31 Putamen


title: BB38: 31 Putamen

value: bb


title: MB38: 31 Putamen


title: CHD16: 25 Corpus Callosum Posterior

source_name: 25 Corpus Callosum Posterior

value: 25 Corpus Callosum Posterior


title: MC37: 32 Caudate

source_name: 32 Caudate

value: 32 Caudate


title: donor49_Mock_day14

value: Mock


title: MA38: 31 Putamen


title: CHD14: 14 Inferior Temporal (BA20)

source_name: 14 Inferior Temporal (BA20)

value: cd

value: 14 Inferior Temporal (BA20)


title: BC37: 32 Caudate


title: BA38: 31 Putamen

value: ba


title: donor50_Infected_day3


title: BB37: 32 Caudate


title: BA39: 26 Internal Capsule

value: 26 Internal Capsule


title: donor50_Mock_day3


title: donor49_Mock_day9


title: CHD12: 2 2ary Auditory (BA22)

source_name: 2  2ary Auditory (BA22)

value: 2  2ary Auditory (BA22)


title: MB37: 32 Caudate


title: CHD11: 18 Ventrolateral Prefrontal (BA44)

source_name: 18 Ventrolateral Prefrontal (BA44)

value: 18 Ventrolateral Prefrontal (BA44)


title: donor49_Mock_day3


title: BA37: 32 Caudate


title: MA37: 32 Caudate


title: CHD10: 10 2ary Visual (BA18/19)

source_name: 10 2ary Visual (BA18/19)

value: 10 2ary Visual (BA18/19)


title: MC36: 20 Entorhinal Cortex

value: mc

value: 20 Entorhinal Cortex


title: BB36: 20 Entorhinal Cortex


title: CHD1: 13 Prefrontal (BA10)

source_name: 13 Prefrontal (BA10)

value: 13 Prefrontal (BA10)


title: donor49_Infected_day9

value: Infected


title: MB36: 20 Entorhinal Cortex

source_name: 20 Entorhinal Cortex

value: mb


title: donor49_Infected_day14

source_name: Primary CD4 positive T-cell


title: BA36: 20 Entorhinal Cortex


title: CHC9: 9 1ary Visual (BA17)

source_name: 9  1ary Visual (BA17)

value: cc

value: 9  1ary Visual (BA17)


title: CHC8: 4 Cingulate Posterior (BA31)

source_name: 4  Cingulate Posterior (BA31)

value: 4  Cingulate Posterior (BA31)


title: BC33: 21 Hippocampus

source_name: 21 Hippocampus

value: bc

value: 21 Hippocampus


title: MA36: 20 Entorhinal Cortex

value: ma


title: MC34: 21 Hippocampus


title: donor49_Infected_day7


title: donor49_Infected_day3


title: donor49_Mock_day7

value: Mock


title: MB34: 21 Hippocampus


title: BA33: 21 Hippocampus

value: ba


title: CHC6: 5 Premotor (BA6)

source_name: 5  Premotor (BA6)

value: 5  Premotor (BA6)


title: CHC7: 3 Precuneus (BA7)

source_name: 3  Precuneus (BA7)

value: 3  Precuneus (BA7)


title: donor14_Mock_day9


title: donor14_Mock_day14


title: BB33: 21 Hippocampus

value: bb


title: CHC5: 11 Cingulate Anterior (BA24)

source_name: 11 Cingulate Anterior (BA24)

value: 11 Cingulate Anterior (BA24)


title: BC32: 15 Insular Posterior Cortex

source_name: 15 Insular Posterior Cortex

value: 15 Insular Posterior Cortex


title: CHD15: 24 Corpus Callosum Anterior

source_name: 24 Corpus Callosum Anterior

value: cd

value: 24 Corpus Callosum Anterior


title: MA34: 21 Hippocampus


title: MC32: 15 Insular Posterior Cortex

value: mc


title: BB32: 15 Insular Posterior Cortex


title: donor14_Mock_day7


title: BC36: 20 Entorhinal Cortex

value: 20 Entorhinal Cortex


title: MB32: 15 Insular Posterior Cortex

value: mb


title: MA32: 15 Insular Posterior Cortex


title: CHC42: 19 Amygdala

source_name: 19 Amygdala

value: cc

value: 19 Amygdala


title: BC31: 7 1ary Auditory (BA41/42)

source_name: 7  1ary Auditory (BA41/42)

value: bc

value: 7  1ary Auditory (BA41/42)


title: donor14_Mock_day3


title: CHC41: 33 Nucleus Accumbens

source_name: 33 Nucleus Accumbens

value: 33 Nucleus Accumbens


title: MC31: 7 1ary Auditory (BA41/42)


title: MB31: 7 1ary Auditory (BA41/42)


title: BB31: 7 1ary Auditory (BA41/42)


title: CHC4: 12 Cingulate Anterior (BA32)

source_name: 12 Cingulate Anterior (BA32)

value: 12 Cingulate Anterior (BA32)


title: donor14_Infected_day7

value: Infected


title: BA31: 7 1ary Auditory (BA41/42)

value: ba


title: CHC39: 26 Internal Capsule

source_name: 26 Internal Capsule

value: 26 Internal Capsule


title: donor14_Infected_day3


title: CHC38: 31 Putamen

source_name: 31 Putamen

value: 31 Putamen


title: BB30: 6 1ary Somatosensory (BA3/1/2)

source_name: 6  1ary Somatosensory (BA3/1/2)

value: bb

value: 6  1ary Somatosensory (BA3/1/2)


title: BC30: 6 1ary Somatosensory (BA3/1/2)


title: MC30: 6 1ary Somatosensory (BA3/1/2)


title: CHC37: 32 Caudate

source_name: 32 Caudate

value: 32 Caudate


title: CHC43: 29 Substantia Nigra

source_name: 29 Substantia Nigra

value: 29 Substantia Nigra


title: BA32: 15 Insular Posterior Cortex

value: 15 Insular Posterior Cortex


title: MA30: 6 1ary Somatosensory (BA3/1/2)

value: ma


title: CHC36: 20 Entorhinal Cortex

source_name: 20 Entorhinal Cortex

value: 20 Entorhinal Cortex


title: donor14_Infected_day14

source_name: Primary CD4 positive T-cell


title: CHC34: 21 Hippocampus

source_name: 21 Hippocampus

value: cc

value: 21 Hippocampus


title: MC29: 8 1ary Motor (BA4)

source_name: 8  1ary Motor (BA4)

value: mc

value: 8  1ary Motor (BA4)


title: BB29: 8 1ary Motor (BA4)


title: BA30: 6 1ary Somatosensory (BA3/1/2)


title: MB30: 6 1ary Somatosensory (BA3/1/2)

value: mb


title: donor14_Infected_day9


title: CHC31: 7 1ary Auditory (BA41/42)

value: 7  1ary Auditory (BA41/42)


title: MB29: 8 1ary Motor (BA4)


title: BA29: 8 1ary Motor (BA4)


title: BC29: 8 1ary Motor (BA4)

value: bc


title: MA31: 7 1ary Auditory (BA41/42)


title: MA29: 8 1ary Motor (BA4)


title: BC28: 1 Supramarginal (BA40)

source_name: 1  Supramarginal (BA40)

value: 1  Supramarginal (BA40)


title: CHC32: 15 Insular Posterior Cortex


title: BB28: 1 Supramarginal (BA40)


title: CHC3: 17 Orbitofrontal (BA11)

source_name: 17 Orbitofrontal (BA11)

value: 17 Orbitofrontal (BA11)


title: MB28: 1 Supramarginal (BA40)


title: BA28: 1 Supramarginal (BA40)

value: ba


title: CHC30: 6 1ary Somatosensory (BA3/1/2)


title: CHC29: 8 1ary Motor (BA4)


title: MC28: 1 Supramarginal (BA40)


title: MA28: 1 Supramarginal (BA40)


title: CHC28: 1 Supramarginal (BA40)


title: MC20: 28 Cerebellar White Matter

source_name: 28 Cerebellar White Matter

value: 28 Cerebellar White Matter


title: CHC22: 30 Cerebellar Grey Matter

source_name: 30 Cerebellar Grey Matter

value: 30 Cerebellar Grey Matter


title: BB22: 30 Cerebellar Grey Matter

value: bb


title: CHC20: 28 Cerebellar White Matter


title: BA22: 30 Cerebellar Grey Matter


title: MA20: 28 Cerebellar White Matter


title: CHC2: 16 Dorsolateral Prefrontal (BA9)

source_name: 16 Dorsolateral Prefrontal (BA9)

value: 16 Dorsolateral Prefrontal (BA9)


title: BB20: 28 Cerebellar White Matter


title: MC19: 30 Cerebellar Grey Matter


title: CHC18: 22 Hypothalamus

source_name: 22 Hypothalamus

value: 22 Hypothalamus


title: MB19: 30 Cerebellar Grey Matter


title: BA20: 28 Cerebellar White Matter


title: CHC17: 23 Thalamus

source_name: 23 Thalamus

value: 23 Thalamus


title: MA19: 30 Cerebellar Grey Matter

value: ma


title: BB18: 22 Hypothalamus


title: BC18: 22 Hypothalamus


title: BC22: 30 Cerebellar Grey Matter


title: CHC16: 25 Corpus Callosum Posterior

source_name: 25 Corpus Callosum Posterior

value: cc

value: 25 Corpus Callosum Posterior


title: MB18: 22 Hypothalamus


title: MB20: 28 Cerebellar White Matter


title: CHC14: 14 Inferior Temporal (BA20)

source_name: 14 Inferior Temporal (BA20)

value: 14 Inferior Temporal (BA20)


title: MA18: 22 Hypothalamus


title: BC17: 23 Thalamus


title: CHC15: 24 Corpus Callosum Anterior

source_name: 24 Corpus Callosum Anterior

value: 24 Corpus Callosum Anterior


title: MC17A: 23 Thalamus

value: mc


title: BA18: 22 Hypothalamus


title: CHC12: 2 2ary Auditory (BA22)

source_name: 2  2ary Auditory (BA22)

value: 2  2ary Auditory (BA22)


title: CHC11: 18 Ventrolateral Prefrontal (BA44)

source_name: 18 Ventrolateral Prefrontal (BA44)

value: 18 Ventrolateral Prefrontal (BA44)


title: BA17: 23 Thalamus


title: MA17: 23 Thalamus


title: BB17: 23 Thalamus


title: BC20: 28 Cerebellar White Matter

value: bc


title: CHC10: 10 2ary Visual (BA18/19)

source_name: 10 2ary Visual (BA18/19)

value: 10 2ary Visual (BA18/19)


title: BC16: 25 Corpus Callosum Posterior


title: MB17: 23 Thalamus

value: mb


title: CHC1: 13 Prefrontal (BA10)

source_name: 13 Prefrontal (BA10)

value: 13 Prefrontal (BA10)


title: MC16B: 25 Corpus Callosum Posterior


title: MC16A: 24 Corpus Callosum Anterior


title: MC18: 22 Hypothalamus


title: BA16: 25 Corpus Callosum Posterior

value: ba


title: BB16: 25 Corpus Callosum Posterior

value: bb


title: CHB9: 9 1ary Visual (BA17)

source_name: 9  1ary Visual (BA17)

value: cb

value: 9  1ary Visual (BA17)


title: MB16B: 25 Corpus Callosum Posterior


title: MB16A: 24 Corpus Callosum Anterior


title: CHB7: 3 Precuneus (BA7)

source_name: 3  Precuneus (BA7)

value: 3  Precuneus (BA7)


title: BB15: 24 Corpus Callosum Anterior


title: CHB6: 5 Premotor (BA6)

source_name: 5  Premotor (BA6)

value: 5  Premotor (BA6)


title: MA16B: 25 Corpus Callosum Posterior

value: ma


title: BA15: 24 Corpus Callosum Anterior


title: MA16A: 24 Corpus Callosum Anterior


title: CHB5: 11 Cingulate Anterior (BA24)

source_name: 11 Cingulate Anterior (BA24)

value: 11 Cingulate Anterior (BA24)


title: BC14: 14 Inferior Temporal (BA20)

value: 14 Inferior Temporal (BA20)


title: MC15: 2 2ary Auditory (BA22)


title: CHB43: 29 Substantia Nigra

source_name: 29 Substantia Nigra

value: 29 Substantia Nigra


title: MB15: 2 2ary Auditory (BA22)


title: BB14: 14 Inferior Temporal (BA20)


title: MA15: 2 2ary Auditory (BA22)


title: CHB41: 33 Nucleus Accumbens

source_name: 33 Nucleus Accumbens

value: 33 Nucleus Accumbens


title: CHB42: 19 Amygdala

source_name: 19 Amygdala

value: 19 Amygdala


title: BC12: 2 2ary Auditory (BA22)

value: 2  2ary Auditory (BA22)


title: CHB8: 4 Cingulate Posterior (BA31)

source_name: 4  Cingulate Posterior (BA31)

value: 4  Cingulate Posterior (BA31)


title: MC14: 18 Ventrolateral Prefrontal (BA44)

value: mc

value: 18 Ventrolateral Prefrontal (BA44)


title: BA14: 14 Inferior Temporal (BA20)


title: CHB4: 12 Cingulate Anterior (BA32)

source_name: 12 Cingulate Anterior (BA32)

value: 12 Cingulate Anterior (BA32)


title: BC15: 24 Corpus Callosum Anterior

value: bc

value: 24 Corpus Callosum Anterior


title: BB12: 2 2ary Auditory (BA22)

value: bb


title: MA14: 18 Ventrolateral Prefrontal (BA44)


title: MB14: 18 Ventrolateral Prefrontal (BA44)

value: mb


title: CHB39: 26 Internal Capsule

source_name: 26 Internal Capsule

value: cb

value: 26 Internal Capsule


title: MC12: 14 Inferior Temporal (BA20)


title: BA12: 2 2ary Auditory (BA22)

value: ba


title: BC11: 18 Ventrolateral Prefrontal (BA44)


title: BB11: 18 Ventrolateral Prefrontal (BA44)


title: CHB36: 20 Entorhinal Cortex

source_name: 20 Entorhinal Cortex

value: 20 Entorhinal Cortex


title: CHB38: 31 Putamen

source_name: 31 Putamen

value: 31 Putamen


title: MB12: 14 Inferior Temporal (BA20)

source_name: 14 Inferior Temporal (BA20)

value: 14 Inferior Temporal (BA20)


title: MA12: 14 Inferior Temporal (BA20)

value: ma


title: CHB34: 21 Hippocampus

source_name: 21 Hippocampus

value: 21 Hippocampus


title: BA11: 18 Ventrolateral Prefrontal (BA44)

source_name: 18 Ventrolateral Prefrontal (BA44)


title: BC10: 10 2ary Visual (BA18/19)

source_name: 10 2ary Visual (BA18/19)

value: 10 2ary Visual (BA18/19)


title: CHB37: 32 Caudate

source_name: 32 Caudate

value: 32 Caudate


title: CHB32: 15 Insular Posterior Cortex

source_name: 15 Insular Posterior Cortex

value: 15 Insular Posterior Cortex


title: MC10: 10 2ary Visual (BA18/19)

value: mc


title: BB10: 10 2ary Visual (BA18/19)


title: MB10: 10 2ary Visual (BA18/19)


title: CHB31: 7 1ary Auditory (BA41/42)

source_name: 7  1ary Auditory (BA41/42)

value: 7  1ary Auditory (BA41/42)


title: BC9: 9 1ary Visual (BA17)

source_name: 9  1ary Visual (BA17)

value: bc

value: 9  1ary Visual (BA17)


title: MC9: 9 1ary Visual (BA17)


title: CHB30: 6 1ary Somatosensory (BA3/1/2)

source_name: 6  1ary Somatosensory (BA3/1/2)

value: 6  1ary Somatosensory (BA3/1/2)


title: MA9: 9 1ary Visual (BA17)


title: CHB29: 8 1ary Motor (BA4)

source_name: 8  1ary Motor (BA4)

value: 8  1ary Motor (BA4)


title: BA9: 9 1ary Visual (BA17)


title: CHB3: 17 Orbitofrontal (BA11)

source_name: 17 Orbitofrontal (BA11)

value: cb

value: 17 Orbitofrontal (BA11)


title: BB9: 9 1ary Visual (BA17)

value: bb


title: BA10: 10 2ary Visual (BA18/19)

value: ba


title: CHB22: 30 Cerebellar Grey Matter

source_name: 30 Cerebellar Grey Matter

value: 30 Cerebellar Grey Matter


title: MB9: 9 1ary Visual (BA17)

value: mb


title: BC8: 4 Cingulate Posterior (BA31)

source_name: 4  Cingulate Posterior (BA31)

value: 4  Cingulate Posterior (BA31)


title: MB8: 3 Precuneus (BA7)

source_name: 3  Precuneus (BA7)

value: 3  Precuneus (BA7)


title: CHB20: 28 Cerebellar White Matter

source_name: 28 Cerebellar White Matter

value: 28 Cerebellar White Matter


title: BB8: 4 Cingulate Posterior (BA31)


title: CHB2: 16 Dorsolateral Prefrontal (BA9)

source_name: 16 Dorsolateral Prefrontal (BA9)

value: 16 Dorsolateral Prefrontal (BA9)


title: CHB18: 22 Hypothalamus

source_name: 22 Hypothalamus

value: 22 Hypothalamus


title: MC7: 4 Cingulate Posterior (BA31)

value: mc


title: CHB17: 23 Thalamus

source_name: 23 Thalamus

value: 23 Thalamus


title: MB7: 4 Cingulate Posterior (BA31)


title: BC7: 3 Precuneus (BA7)

value: bc


title: BA7: 3 Precuneus (BA7)


title: CHB28: 1 Supramarginal (BA40)

source_name: 1  Supramarginal (BA40)

value: 1  Supramarginal (BA40)


title: MA7: 4 Cingulate Posterior (BA31)

value: ma


title: BB7: 3 Precuneus (BA7)


title: MC6: 5 Premotor (BA6)

source_name: 5  Premotor (BA6)

value: 5  Premotor (BA6)


title: CHB16: 25 Corpus Callosum Posterior

source_name: 25 Corpus Callosum Posterior

value: cb

value: 25 Corpus Callosum Posterior


title: MC8: 3 Precuneus (BA7)


title: CHB14: 14 Inferior Temporal (BA20)

source_name: 14 Inferior Temporal (BA20)

value: 14 Inferior Temporal (BA20)


title: MA6: 5 Premotor (BA6)


title: MB6: 5 Premotor (BA6)


title: BC6: 5 Premotor (BA6)


title: MC5: 11 Cingulate Anterior (BA24)

source_name: 11 Cingulate Anterior (BA24)

value: 11 Cingulate Anterior (BA24)


title: 4T07-Six2 cells replicate 2

city: Aurora

first: Michael

last: Oliphant

state: CO

department: Pharamacology

institute: University of Colorado Anschutz Medical Campus

description:
4T07_Six2_rep2
total RNA from 4T07-Six2 cells

data_processing:
Signal processing, base-calling, and quality scoring was carried out automatically by the HiSeq 2500 Real-Time Analysis (RTA) software (Illumina)
Demultiplexing and conversion to FASTQ format was carried out using bcl2fastq (v1.8.4, Illumina)
FASTQC (v0.11.5) was used to assess sequence data quality
Low quality bases (Q <10) were trimmed from the 3' end of reads and short reads (<30 nt after trimming) and adaptor sequences were removed using the fastqc-mcf tool from ea-utils
FastQ Screen (v0.9.1) was used to check for common sequencing contaminants
Alignment to the mouse reference genome (GRCm38/mm10) was carried out using TopHat2 (v2.1.1, --b2-sensitive --keep-fasta-order --no-coverage-search --max-multihits 10 --library-type fr-firststrand) with the UCSC mm10 GTF annotation file provided in the iGenomes UCSC mm10 bundle (https://support.illumina.com/sequencing/sequencing_software/igenome.html)
Aligned reads were then filtered to remove low quality mapped reads (MAPQ < 10) using SAMtools (v1.5)
Reads were coordinate sorted and duplicates were marked using Picard (v2.9.4)
Quality assessment of final mapped reads was performed using RSeQC (v2.6.4)
Raw gene-level count data was generated using HTSeq (v0.8.0) with the following options (--stranded=reverse –minaqual=10 –type=exon –idattr=gene --mode=intersection-nonempty) using the GTF provided in the UCSC mm10-based igenomes bundle (https://support.illumina.com/sequencing/sequencing_software/igenome.html)
Genome_build: GRCm38/mm10; Bowtie2 index and GTF from iGenomes UCSC mm10 bundle (https://support.illumina.com/sequencing/sequencing_software/igenome.html)
Supplementary_files_format_and_content: Tab delimited text file containing raw gene-level counts for each sample

organism: Mus musculus

growth_protocol: 4T07 CTL and Six2 cells were grown in 10cm dishes to approximately 75-80% confluency in DMEM/High Glucose supplemented with 10% calf serum, Non-essential amino acids (NEAA), L-Glutamine at 37ºC/5% CO2

extract_protocol:
At 48h post plating, cells were washed with 1X cold PBS and scraped into RLT Plus Lysis Buffer. RNA was then isolated using the Qiagen Rneasy Plus Kit per manufacturer's instructions.
Libraries for Illumina sequencing were prepared using a TruSeq Stranded mRNA Library Prep Kit (Illumina), according to the manufacturer’s instructions.

tag: strain

tag: cell line

value: murine mammary carcinoma

tag: genotype

value: Six2 Overexpression

tag: culture conditions

value: 37°C, 5% CO2, 2 days


title: BB6: 5 Premotor (BA6)

value: bb


title: CHB12: 2 2ary Auditory (BA22)

source_name: 2  2ary Auditory (BA22)

value: 2  2ary Auditory (BA22)


title: BA8: 4 Cingulate Posterior (BA31)

value: ba

value: 4  Cingulate Posterior (BA31)


title: CHB11: 18 Ventrolateral Prefrontal (BA44)

source_name: 18 Ventrolateral Prefrontal (BA44)

value: 18 Ventrolateral Prefrontal (BA44)


title: MA8: 3 Precuneus (BA7)

value: 3  Precuneus (BA7)


title: BC5: 11 Cingulate Anterior (BA24)

value: bc


title: MB5: 11 Cingulate Anterior (BA24)

value: mb


title: 4T07-Six2 cells replicate 1

description:
4T07_Six2_rep1
total RNA from 4T07-Six2 cells


title: 4T07-CTL cells replicate 2

description:
4T07_CTL_rep2
total RNA from 4T07-CTL cells

value: control


title: CHB10: 10 2ary Visual (BA18/19)

source_name: 10 2ary Visual (BA18/19)

value: 10 2ary Visual (BA18/19)


title: MA5: 11 Cingulate Anterior (BA24)

value: ma


title: 4T07-CTL cells replicate 1

description:
4T07_CTL_rep1
total RNA from 4T07-CTL cells


title: CHB15: 24 Corpus Callosum Anterior

source_name: 24 Corpus Callosum Anterior

value: cb

value: 24 Corpus Callosum Anterior


title: BA5: 11 Cingulate Anterior (BA24)

value: 11 Cingulate Anterior (BA24)


title: MC4: 12 Cingulate Anterior (BA32)

source_name: 12 Cingulate Anterior (BA32)

value: mc

value: 12 Cingulate Anterior (BA32)


title: CHB1: 13 Prefrontal (BA10)

source_name: 13 Prefrontal (BA10)

value: 13 Prefrontal (BA10)


title: BC4: 12 Cingulate Anterior (BA32)


title: MB4: 12 Cingulate Anterior (BA32)


title: MA4: 12 Cingulate Anterior (BA32)


title: BA6: 5 Premotor (BA6)

value: 5  Premotor (BA6)


title: BB4: 12 Cingulate Anterior (BA32)

value: bb


title: HD2

city: Shanghai

first: Weiqiang

last: Cao

state: Shanghai

address: 130 Meilong Road

country: China

institute: East China University of  Science and Technology

data_processing:
Illumina Casava1.7 software used for basecalling.
The clean reads were produced after the raw reads were filtered to the following criteria: remove reads with sequence adaptors; remove reads with more than 10% ‘N’ bases; and remove low-quality reads, which have more than 50% QA ≤ 10 bases.
Clean reads were then aligned to the reference genome and no more than three mismatches were allowed in the alignment for each read. Reads that could be uniquely mapped to a gene were used to calculate the expression level. The gene expression level was quantified using RSEM (Li and Dewey 2011).
The formula to calculate FPKM (Fragments Per Kilobase of exon model per Million mapped fragments) value is as follows:FPKM = 106C/(NL/103), in which FPKM (A) is the expression level of gene A. C is counts of fragments that uniquely mapped to the gene A. N is total counts of fragments that uniquely mapped to the reference genes. L is the base counts of the coding region of gene A.
Genome_build: A. niger strain CBS 513.88 (Genbank ID:GCA_000002855.2)
Supplementary_files_format_and_content: excel files including FPKM values for each Sample

organism: Aspergillus niger

source_name: Aspergillus niger

growth_protocol: A 5 L fermenter with an electronic balance was used for the fed-batch cultivations with the agitation rate at 375 rpm and the aeration at 1 vvm. During the cultivation, the overpressure was maintained at 0.05 MPa and the temperature was at 34 ºC .The broth pH was maintained at 4.5.

extract_protocol:
After sampling at 18h,24h,42h and 66h, the broth was immediately frozen in liquid nitrogen and stored at -80  ºC for RNA extraction.
RNA libraries were prepared for sequencing using standard Illumina protocols

treatment_protocol: oxygen limitation fermentation

tag: growth phase

value: oxygen limited late phase


title: HC3

value: oxygen limited middle phase


title: MC3: 17 Orbitofrontal (BA11)

source_name: 17 Orbitofrontal (BA11)

value: 17 Orbitofrontal (BA11)


title: HD1


title: HC2


title: MB3: 17 Orbitofrontal (BA11)


title: HC1


title: HB2

value: transitional phase/oxygen limited early stage


title: BA4: 12 Cingulate Anterior (BA32)

value: ba


title: BB3: 17 Orbitofrontal (BA11)


title: BA3: 17 Orbitofrontal (BA11)


title: BC3: 17 Orbitofrontal (BA11)

value: bc


title: HB3


title: BB5: 11 Cingulate Anterior (BA24)


title: MC2: 16 Dorsolateral Prefrontal (BA9)

value: 16 Dorsolateral Prefrontal (BA9)


title: BC2: 16 Dorsolateral Prefrontal (BA9)


title: MA3: 17 Orbitofrontal (BA11)


title: MB2: 16 Dorsolateral Prefrontal (BA9)

value: mb


title: HB1


title: BB2: 16 Dorsolateral Prefrontal (BA9)


title: MA2: 16 Dorsolateral Prefrontal (BA9)

value: ma


title: BA2: 16 Dorsolateral Prefrontal (BA9)


title: BC1: 13 Prefrontal (BA10)


title: HA2

value: exponential phase


title: MB1: 13 Prefrontal (BA10)


title: HA1


title: BB1: 13 Prefrontal (BA10)


title: MA1: 13 Prefrontal (BA10)


title: EV cells of HCT-116 cells

status: Public on Jan 29 2020

city: Suita

first: Daisuke

last: Okuzaki

state: Osaka

address: Yamadaoka 3-1

department: Immunology Frontier Research Center

country: Japan

institute: Osaka univ.

description: empty vector-transfected control (EV) cells of HCT-116 cells

data_processing:
Sequencing was performed on an Illumina HiSeq 2500 platform in a 75-base single-end mode.
Illumina Casava1.8.2 software used for basecalling.
Sequenced reads were mapped to the human reference genome sequences (hg19) using TopHat v2.0.13 in combination with Bowtie2 ver. 2.2.3 and SAMtools ver. 0.1.19.
The fragments per kilobase of exon per million mapped fragments (FPKMs) was calculated using Cuffnorm version 2.2.1.
Genome_build: hg19
Supplementary_files_format_and_content: FPKM

extract_protocol:
Total RNA was extracted from EV cells and SRR-KO cells of DLD-1 cells and HCT116 cells using the miRNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol.
Library preparation was performed using a TruSeq stranded mRNA sample prep kit (Illumina, San Diego, CA) according to the manufacturer’s instructions.


title: BA1: 13 Prefrontal (BA10)


title: SRR-KO of HCT-116 cells

description: SRR knockout (SRR-KO) cells of HCT-116 cells


title: SOX2_PAR-CLIP_rep1

city: Guangzhou

first: Yiwei

last: Lai

state: Guangdong

address: 190 Kai Yuan Avenue, Science Park

department: South China Institute for Stem Cell Biology and Regenerative Medicine Key Laboratory of Regenerative Biology, CAS

institute: Guangzhou Institutes of Biomedicine and Health,Chinese Academy of Sciences

data_processing:
Illumina bcl2fastq2.17 software was used for basecalling.
Adapter sequences and reads shorter than 15 bp were eliminated, identical reads were collapsed using FASTX-Toolkit.
Reads were aligned to hg19 genome assembly using bowtie version 1.1.2 with the following configuration, -p 8 -v 2 -m 1 -f.
Aligned reads were used for peak calling using PARalyzer v1.5, requiring a minimum cluster size of 15 nucleotides, a minimum conversion count of 2 T > C conversion and 5 minimum read counts for cluster inclusion.
Peaks were used for motifs finding with MEME suite.
Genome_build: hg19
Supplementary_files_format_and_content: Bed files were extracted  according to PARalyzer output. Bed files including the information of chromosome, start, end, peak ID, read count, strand.

source_name: HEK293T cells

growth_protocol: HEK293T cells were cultured in DMEM/HIGH GLUCOSE (Hyclone) supplemented with 10% FBS (Biowest), nonessential amino acids (Hyclone), penicillin/streptomycin (Invitrogen), and GlutaMax (Invitrogen)

extract_protocol:
Samples were treated with proteinase K (Merck), and RNA was isolated using phenol/chloroform extraction.
cDNAs were synthesized with SuperScript III (#18080093) and amplified with a 18 cycles PCR. The PCR products were selected based on size using from PAGE gels according to instructions of TruSeq® Small RNA Sample Prep Kit (Illumina, Cat#15019892).
The purified library products were evaluated using the Agilent 2200 TapeStation and diluted to 10 pM for cluster generation and high-throughput sequencing (50 bp single end) on HiSeq 2500 (Illumina).

treatment_protocol: HEK293T cells were transfected with plasmids expressing FLAG-tagged SOX2 protein with 1 μg/μl Polyethylenimine (Polyscience). 24 hours after transfection, cells were treated with 0.2 mM 4-thiouridine (4SU) for another 16 hours. Cells were then crosslinked with 0.4 J/cm2 of 365 nm UV light, scraped off with a rubber scraper in 2 ml PBS per plate, and collected by centrifugation at 500 g for 5 mins at 4°C. Crosslinked cell pellets were resuspended in 300 μl (for each plate) NP40 lysis buffer (50 mM HEPES pH 7.5,150 mM KCl, 2 mM EDTA, 1 mM NaF, 0.5% NP40, 0.5 mM DTT, and protease inhibitor cocktail (Roche)) and incubated on ice for 10 mins. Lysates were homogenized by 0.4 mm syringe needle and centrifuged at 13,000 g for 15 mins at 4°C. The supernatant was digested with RNase T1 (Sigma) at a final concentration of 1 U/ml at 22°C for 15 mins and cooled for 5 mins on ice immediately after digestion. Cell lysates were then added to magnetic beads (Sigma) conjugated with FLAG antibodies (10 μl for each plate) and incubated for 2 hours at 4°C. After that, magnetic beads were collected with a magnetic particle collector (Invitrogen). Beads were washed 3 times with 1 ml of washing buffer (50 mM HEPES-KOH pH 7.5, 300 mM KCl, 0.05% NP40, 0.5 mM DTT, and protease inhibitor cocktail (Roche)) at 4°C, and resuspended in washing buffer (10 μl for each plate) with RNase T1 at a final concentration of 10 U/ml for 15 mins at 22°C, followed by immediate cooling for 5 mins on ice. Beads were washed 3 times with 1 ml of high-salt washing buffer (50 mM HEPES-KOH pH 7.5, 500 mM KCl, 0.05% NP40, 0.5 mM DTT, and protease inhibitor cocktail (Roche)) at 4°C. Beads were resuspended in dephosphorylation buffer (10 μl for each plate, 50 mM Tris-HCl, pH 7.9, 100 mM NaCl, 10 mM MgCl2, and 1 mM DTT) supplemented with calf intestinal alkaline phosphatase (CIAP, NEB) at a final concentration of 0.5 U/ml, and the suspension was incubated for 10 mins at 37°C. After incubation, beads were washed twice with 1 ml of phosphatase wash buffer (50 mM Tris-HCl, pH 7.5, 20 mM EGTA, and 0.5% NP40) and twice with 1 ml of T4 polynucleotide kinase (PNK) buffer without DTT (50 mM Tris-HCl pH 7.5, 50 mM NaCl, and 10 mM MgCl2). The RNAs co-immunoprecipitated proteins were treated with 1 U/μl T4 PNK (NEB) for 60 mins at 37°C. After that, the samples were washed, eluted and run with Novex Bis-Tris 4-12% (Invitrogen) precast PAGE gel in MOPS buffer (Invitrogen). RNA-protein complexes were then transferred onto an NC membrane, which was cut according to the size visualized by Fusion Solos System from Vilber Lourmat on a parallel experiment for PAR-CLIP-biotin chemiluminescent nucleic acid detection. The membrane was cut into small slices for RNA extraction.

value: Derived from human embryonic kidney cells


title: SOX2_PAR-CLIP_rep2


title: Micro-C_CTCF_noauxin_rep2

city: Berkeley

first: Robert

last: Tjian

address: LKS

institute: UC Berkeley/HHMI

description: Homozygote cell line with endogenously tagged CTCF; DMSO treated for 6 hr

library_source: genomic

data_processing:
Library strategy: Micro-C
Base-calling performed with CASAVA by the Vincent J. Coates Genomics Sequencing Laboratory at UC Berkeley
allValidPairs were generated by HiC-Pro package (>2.10 version)
mcool files were converted from allValidPairs by Cooler package
hic file were converted from allValidPairs by Juicer tools
Genome_build: mm10
Supplementary_files_format_and_content: allValidParis file contains 1) read name, 2) chromosome of first locus, 3) read position of first locus,  4) strand of first locus, 5) chromosome of second locus, 6) read position of second locus, 7) strand of second locus, 8) DNA fragment lengh, 9) name of first restriction fragment, 10) name of second restiction fragment, 11) mapping quality of first read, 12) mapping quality of second read.
Supplementary_files_format_and_content: mcool file contains all valid Micro-C pairs stored in HDF5 format with mutiple resolutions
Supplementary_files_format_and_content: hic file contains all valid Micro-C pairs with mutiple resolutions

molecule: genomic DNA

source_name: embryonic stem cells

growth_protocol: JM8.N4 mouse embryonic stem cells were grown on plates pre-coated with a 0.1% autoclaved gelatin solution (Sigma-Aldrich, G9391) under feeder free condition in knock-out DMEM with 15% FBS and LIF (full recipe: 500 mL DMEM (ThermoFisher#10829018), 6 mL MEM NEAA (ThermoFisher #11140050), 6 mL GlutaMax (ThermoFisher#35050061), 5 mL Penicillin-streptomycin (ThermoFisher #15140122), 4.6 μL 2-mercapoethanol (Sigma-Aldrich M3148), 90 mL fetal bovine serum (HyClone FBS SH30910.03 lot #AXJ47554)). mES cells were fed by replacing half the medium with fresh medium daily and passaged every two days by trypsinization.

extract_protocol:
Micro-C protocol was performed as the following steps: 1) digest crosslinked chromatin by MNase; 2) Repair fragment ends with biotin-dNTP; 3) Proximal ligation and purge unligated ends; 4) Purify ligated dinucleosomal DNA.
Purified DNA with biotin-dNTPs was captured by Dynabeads® MyOne™ Streptavidin C1. Standard library preparation protocol including end-repair, A-tailing, and adapter ligation was performed on beads with individual enzymes purchased from Lucigen or NEBnext UltraII kit. Sequencing library was amplified by Kapa HiFi PCR enzyme with lowest possible cycles to reduce PCR duplicates. Sequencing library was sequenced paired-end 50x50 in Illumina HiSeq 4000 sequencer.

treatment_protocol: Cells were crosslinked by 1% formaldehyde for 10 min at room temperature. Formaldehyde reaction was quenched by 0.75M Tris. After two times of PBS washes, cells were then crosslinked by 3mM DSG for 45 min at room temperature. After two times of PBS washed, cell pellets were snap frozen or subjected to the following protocols.

value: AID-mCTCF

tag: ctcf protein

value: wild type


title: Micro-C_CTCF_auxin_6hour_rep1

description: Homozygote cell line with endogenously tagged CTCF; 500nM Auxin treated for 12 hr


title: UCC_EU2015_8

city: NEW YORK

first: WENHUO

last: HU

email: huwenhuo@gmail.com, huw@mskcc.org

state: NEW YORK

address: 1275 YORK AVENUE

department: HOPP

institute: MEMORIAL SLOAN KETTERING CANCER CENTER

data_processing:
RSEM
tximport
DESeq2
Genome_build: GRCh38
Supplementary_files_format_and_content: normalized reads count

source_name: Upper Tract Urothelial Cell Carcinoma

extract_protocol:
Qiagen Rneasy mini Kit
TruSeq RNA Library Prep Kit V2

tag: cell type

value: Upper Tract Urothelial Cell Carcinoma


title: UCC_EU2015_68


title: Micro-C_CTCF_auxin_6hour_rep2


title: Micro-C_CTCF_noauxin_rep1


title: UCC_EU2015_67


title: UCC_EU2015_7


title: UCC_EU2015_9


title: UCC_EU2015_65


title: UCC_EU2015_64


title: HA3


title: UCC_EU2015_62


title: UCC_EU2015_63


title: UCC_EU2015_61


title: MC1: 13 Prefrontal (BA10)

value: mc

value: 13 Prefrontal (BA10)


title: EV cells of DLD-1 cells

description: empty vector-transfected control (EV) cells of DLD-1 cells

value: DLD-1 cells


title: UCC_EU2015_59


title: UCC_EU2015_58


title: UCC_EU2015_6


title: UCC_EU2015_57


title: UCC_EU2015_56


title: ROW1505A20_20

type: genomic

city: Melbourne

first: TANJINA

last: KADER

state: VIC

address: 305 Grattan Street

department: Cancer Genomics

country: Australia

institute: The Sir Peter MacCallum Cancer Centre

hyb_protocol: standard affymetrix protocol

scan_protocol: standard affymetrix protocol

data_processing: Copy number values were obtained against a baseline of normal DNA samples. Raw data is only available for the single file run on OncoScan v3. The rest are available in 2 matrix files, and have slightly different numbers of probes. Each sample has 5 columns: Copy A, Copy B, Copy number, Allele ratio and Allelic difference. P1 and P2 was run on OncoScan v2.

label: biotin

source_name: Pure papilloma FFPE tissue

label_protocol: standard affymetrix protocol

extract_protocol: up to 40x 10 micron FFPE sections were needle microdissected for epithelial cells. Total genomic DNA was isolated using the DNeasy Blood and Tissue Kit (Qiagen) using a modified protocol: Wu L, Patten N, Yamashiro CT, Chui B (2002) Extraction and amplification of DNA from formalin-fixed, paraffin-embedded tissues. Appl Immunohistochem Mol Morphol 10 (3):269-274

treatment_protocol: retrospective cases received standard care

tag: histopath subtype

value: atypical papillary leison

tag: p63 status

value: Positive

tag: er status

tag: her2 copy number status

value: Negative


title: SRR-KO of DLD-1 cells

description: SRR knockout (SRR-KO) cells of DLD-1 cells


title: UCC_EU2015_55


title: UCC_EU2015_53


title: UCC_EU2015_54


title: UCC_EU2015_52


title: UCC_EU2015_5


title: UCC_EU2015_50


title: UCC_EU2015_49


title: UCC_EU2015_48


title: UCC_EU2015_47


title: UCC_EU2015_45


title: UCC_EU2015_43


title: UCC_EU2015_46


title: UCC_EU2015_39


title: UCC_EU2015_36


title: UCC_EU2015_37


title: UCC_EU2015_60


title: UCC_EU2015_35


title: UCC_EU2015_4


title: UCC_EU2015_33


title: UCC_EU2015_32


title: UCC_EU2015_30


title: UCC_EU2015_3


title: UCC_EU2015_28


title: UCC_EU2015_27


title: UCC_EU2015_26


title: UCC_EU2015_21


title: UCC_EU2015_31


title: UCC_EU2015_24


title: UCC_EU2015_25


title: UCC_EU2015_19


title: UCC_EU2015_42


title: UCC_EU2015_20


title: UCC_EU2015_17


title: UCC_EU2015_13


title: UCC_EU2015_11


title: UCC_EU2015_38


title: UCC_EU2015_18


title: UCC_EU2015_1


title: UCC_EU2015_15


title: UCC_EU2015_10


title: UCC5_primary


title: UCC6_primary


title: UCC_EU2015_2


title: UCC30_primary


title: UCC1_primary


title: UCC17_primary


title: UCC3_met_abdominal_wall


title: UCC20_primary


title: UCC22_primary


title: UCC14_primary


title: UCC34_primary


title: UCC42_primary


title: UCC46_primary


title: UCC28_primary


title: UCC47_primary


title: UCC40_primary


title: UCC21_primary


title: UCC13_primary


title: UCC37_primary


title: UCC25_primary


title: E55m_MS117_106 (SingleCell605)

city: Kyoto

first: Yukihiro

last: Yabuta

state: Kyoto

address: Yoshida-Konoe-cho, Sakyo-ku

department: Anatomy and Cell Biology

institute: Kyoto University, Graduate school of medicine

description: cDNA from embryonic gonad was amplified and its annotations was evaluated by qPCR as germ cells

data_processing:
library strategy: SC3-seq
Samples were sequenced on NextSeq and SOLiD sequencers with the read length are 76 and 50 bases.
All the reads were surveyed and the adaptor or the poly-A sequences were trimmed by cutadapt-1.3 with options "-e 0.1 -q 20 -n 2 -O 1 -m 30 -a CTCGAGGGCGCGCCGGATCC -g CTCGAGGGCGCGCCGGATCC -a AAAAAAAAAAAAAAAAAAAA -a TTTTTTTTTTTTTTTTTTTT". The trimmed reads with less than 30 bp were discarded.
Untrimmed and trimmed reads of 30 bp or longer were mapped onto the Cynomolgus monkey genome Macfac5.0 and the ERCC spike-in RNA sequences with tophat-1.4.1/bowtie1.0.1 with the “—no-coverage-search” option.
Mapped reads on the genome and the ERCC were separated, and the reads on the genome were converted into the expression levels by cufflinks-2.2.0 using the “—compatible-hits-norm”,  “—max-mle-iterations 50000", “—no-length-correction” and “—library-type fr-secondstrand” options and macfas5.0 reference gene annotations with extended TTSs. For the reference gene annotations used in cufflinks, we extended the TTSs of the reference genes up to 10 kb downstream to correctly estimate the expression levels of genes whose transcripts are longer than the reference toward the 3 prime.
Genome_build: macfas5.0
Supplementary_files_format_and_content: tab-delimited text file includes RPM values for each Sample

organism: Macaca fascicularis

source_name: Embryonic gonadal germ cell from E55 embryo

growth_protocol:
For cultivation of CMK6 and CMK9 cyESCs, they were cultured with conventional hESC medium [DMEM/F12 supplemented with 20% (vol/vol) of KSR, 1 mM of sodium pyruvate, 2 mM of GlutaMax, 0.1 mM of non-essential amino acids, 0.1 mM of 2-mercaptoethanol, 1000 U/ml of ESGRO mouse LIF, and 4 ng/ml of recombinant human bFGF] on mouse embryonic feeders (MEFs).
For cultivation in 656BTAG42 cells under undifferentiated state, it was cultured in AITS medium containing 20ng/mL bFGF and 2.5nM IWR1

extract_protocol:
For single cell isolation from cy pre-implantation embryos, a whole embryo was incubated with 0.25% trypsin/PBS  for around 10 min at 37℃, then dissociated into single cells by repeated pipetting, and dispersed in 0.1 mg/ml of PVA/PBS  for preparation of single-cell cDNAs.
For single cell isolation from cy post-implantation embryos, the implantation site was dissected out from the uterus and the embryonic fragment containing the epiblast (EPI), amnion, hypoblast, and yolk sac endoderm was isolated manually. The fragment was incubated with 0.25% trypsin/PBS for around 10 min at 37℃, then dissociated into single cells by repeated pipetting, and dispersed in 0.1 mg/ml of PVA/PBS.
For single cell isolation from cyESCs, cells were first detached as clumps with CTK solution, incubated in 0.25% trypsin/PBS (Sigma-Aldrich) for around 10 min at 37℃, and dispersed into single cells in 1% (vol/vol) KSR/PBS containing 10 M of the ROCK inhibitor Y-27632. Cells under feeder-free condition were directly incubated in TrypLE Select for around 5 min at 37℃, and dispersed into single cells in 1% (vol/vol) KSR/PBS containing 10 uM of the ROCK inhibitor Y-27632.
For single cell isolation from cyPGCLCs, the cells dissociated with TryPLE select were collected by cell sorting.
For SC3-seq analysis, cDNA synthesis / amplification from single cells and library construction from the cDNAs were performed as described previously [Nakamura et al. 2015, NAR, 43, e60].

treatment_protocol: For cyPGCLCs, undifferentiated ESCs were dissociated and replated to low-adherent V-bottom plates to form aggregates with BMP4 (200 ng/ml), SCF (100 ng/ml), EGF (50 ng/ml), LIF(1000U/ml), and Y27632 (10µM) in GMEM containing 15% KSR or Advanced RPMI1640 containing B27, until the harvest day.

value: not applicable

value: Embryonic gonad

tag: developmental stage


title: UCC31_primary


title: UCC51_primary


title: UCC8_primary_pelvis


title: E51f_MS117_081 (SingleCell602)

source_name: Embryonic gonadal germ cell from E51 embryo


title: E51f_MS117_080 (SingleCell601)


title: E51f_MS132_069 (SingleCell603)


title: E51f_MS117_062 (SingleCell598)


title: E51f_MS117_077 (SingleCell599)


title: E51f_MS117_078 (SingleCell600)


title: E51f_MS132_079 (SingleCell604)


title: E50f_MS132_215 (SingleCell597)

source_name: Embryonic gonadal germ cell from E50 embryo


title: E50f_MS117_076 (SingleCell596)


title: E50f_MS117_072 (SingleCell593)


title: E36m_MS117_100 (SingleCell582)

source_name: Embryonic gonadal germ cell from E36 embryo


title: E36m_MS117_103 (SingleCell585)


title: E43f_MS117_061 (SingleCell587)

source_name: Embryonic gonadal germ cell from E43 embryo


title: E36m_MS117_102 (SingleCell584)


title: E43f_MS117_063 (SingleCell588)


title: E43f_MS117_064 (SingleCell589)


title: E43f_MS117_068 (SingleCell590)


title: E36m_MS117_097 (SingleCell581)


title: E50f_MS117_075 (SingleCell595)


title: E50f_MS117_073 (SingleCell594)


title: E20_MS126_087 (SingleCell578)

description: Cynomolgus Embryo at E20

source_name: Cynomolgus Embryo at E20

value: embryo


title: E43f_MS117_060 (SingleCell586)


title: E20_MS117_092 (SingleCell577)

description: Cynomolgus Embryo at E20


title: E20_MS126_089 (SingleCell580)

description: Cynomolgus Embryo at E20


title: E47m_MS117_105 (SingleCell592)

source_name: Embryonic gonadal germ cell from E47 embryo


title: E36m_MS117_101 (SingleCell583)


title: UCC_EU2015_14


title: E17_MS127_139 (SingleCell572)

description: Cynomolgus Embryo at E17

source_name: Cynomolgus Embryo at E17


title: E17_MS127_142 (SingleCell575)

description: Cynomolgus Embryo at E17


title: E17_MS127_138 (SingleCell571)

description: Cynomolgus Embryo at E17


title: CRAC_rRNA_control_15

city: Basel

first: Alex

middle: Charles

last: Tuck

address: Maulbeerstrasse 66

country: Switzerland

institute: Friedrich Miescher Institute for Biomedical Research

description:
in-line barcode (present at 5' end of CRAC reads): NNNNACTCAGC
rRNA analysis samples: sample used only for rRNA CRAC analysis

data_processing:
Library strategy: CRAC (long protocol)
CRAC analysis of binding to rRNA: To determine specific binding sites on ribosomes from CRAC data, additional datasets with low global coverage but sufficient rRNA coverage were used, as well as a panel of control CRAC experiments analysing unrelated proteins or untagged/non-crosslinked cells. Samples used only for rRNA analysis are indicated in the "rRNA analysis" column
processed data files format and content: For CRAC, bigwig files are provided. Values are reads per million, and replicates for the same protein are pooled.
processed data files format and content: For RNA-seq, differential expression was analysed using DESeq2 and values are provided in Supplementary_table_3.txt. This table also contains some processed CRAC counts, that were used in conjunction with the RNA-seq analysis to elucidate the functions of SKIV2L and AVEN. Supplementary_table_3.txt contains the following columns:
processed data files format and content: Column 1: Gene ID.
processed data files format and content: Columns 2-3: Half-life analysis from PMID: 30456373.
processed data files format and content: Columns 4-7: Relative CRAC counts for Aven, Mtr4, Xrn1 and Skiv2l. For each gene, the APPRIS principal isoform is used for counting (picking one at random in case there are multiple).
processed data files format and content: Columns 8-9: tSNE coordinates based on relative CRAC counts for Mtr4, Xrn1 and Skiv2l, used to arrange transcripts by their relative binding to these three factors (which reflect RNA degradation by the three major mammalian decay pathways).
processed data files format and content: Column 10: Transcript ID used for CRAC counting.
processed data files format and content: Column 11: Gene name.
processed data files format and content: Columns 12-14: CDS and transcript length details.
processed data files format and content: Columns 15-18: RNA-seq RPKM values for Mtr4, Xrn1 and Skiv2l cell lines used for CRAC (3xFLAG-Avi-tagged lines).
processed data files format and content: Columns 19-21: Relative CRAC counts for Mtr4, Xrn1 and Skiv2l normalized to RNA-seq.
processed data files format and content: Columns 22-30: DESeq2 base mean, log-fold change and adjusted p value for Skiv2lfl/fl knock out after 2, 4 or 6 days of 4OHT addition, versus untreated.
processed data files format and content: Columns 31-33: DESeq2 base mean, log-fold change and adjusted p value for Aven-/- versus wild-type mESCs.
processed data files format and content: Columns 34-39: DESeq2 base mean, log-fold change and adjusted p value for Skiv2lfl/fl Aven-/- knock out after 2 or 4 days of 4OHT addition, versus untreated.
Genome_build: mm10

source_name: mouse embryonic stem cell

growth_protocol: Cells were cultured on gelatin-coated dishes in serum+LIF medium (DMEM, 0.1 mM non-essential amino acids, 1 mM sodium pyruvate, 2 mM L-glutamine, 15 % (v/v) fetal bovine serum, 0.1 mM beta-mercaptoethanol, 50 μg/ml penicillin, 80 μg/ml streptomycin and home-made LIF conditioned medium)

extract_protocol:
mESCs were grown in 2x 15-cm dishes to ~80 % confluency, dishes washed 2x with PBS, the PBS removed, then cells crosslinked on ice (with dishes facing up) in a Stratagene Stratalinker 2400 (400 mJ cm?2). Cells were lysed by incubating with 5 ml of either TN150+NP40 (50 mM Tris-HCl pH 7.8, 150 mM NaCl, 0.5 % Nonidet P40 substitute and 1x cOmplete Protease Inhibitor Cocktail), or of RIPA (50 mM Tris-HCl pH 7.8, 300 mM NaCl, 1.0 % Nonidet P40 substitute, 0.1 % SDS, 10 % (v/v) glycerol, 0.5 % sodium deoxycholate, 1 mM beta-mercaptoethanol, 1x cOmplete Protease Inhibitor Cocktail), as indicated. The harsher buffer (RIPA) was initially used to ensure complete extraction, but as this can reduce FLAG binding we later switched to a milder buffer (TN150), which did not affect library content. The cells were further disrupted using a cell scraper then lysates collected and centrifuged (6500 xg for 20 min at 4 °C). Supernatants were frozen in liquid nitrogen and stored at -80 °C.
Lysates were thawed on ice and incubated with 100 µL anti-FLAG M2 magnetic beads overnight. The supernatant was discarded and beads washed 3x with 1 ml TN150 (50 mM Tris-HCl pH 7.8, 150 mM NaCl, 0.1 % Nonidet P40 substitute). Protein:RNA complexes were eluted by incubating beads in 1.5 ml TN150 supplemented with 5 mM beta-mercaptoethanol and 0.3 mg/ml 3xFLAG peptide, rotating at 4 °C for 2 hr. The eluate was then incubated with 50 µL Dynabeads M-280 Streptavidin, rotating at 4 °C overnight. Beads were washed 2x in TN600 (50 mM Tris-HCl pH 7.8, 600 mM NaCl, 0.1 % Nonidet P40 substitute, 5 mM beta-mercaptoethanol) and 2x in TN150 supplemented with 5 mM beta-mercaptoethanol. RNA was fragmented by incubating beads in 500 ?L TN150 supplemented with 5 mM beta-mercaptoethanol and 1 ?L of 0.1 U diluted RNace-IT. After 4 min at 37 °C, the RNases were denatured by replacing the solution with 400 ?L WBI (50 mM Tris-HCl pH 7.8, 300 mM NaCl, 0.1 % Nonidet P40 substitute, 5 mM beta-mercaptoethanol and 4.0 M guanidine hydrochloride). The beads were washed 2x in WBI, then 3x in 400 ?L 1xPNK (50 mM Tris-HCl pH 7.8, 10 mM MgCl2, 0.5 % Nonidet P40 substitute, 5 mM beta-mercaptoethanol).
The following four enzymatic reactions were then performed in 80 ?L 1xPNK buffer (omitting the Nonidet P40 substitute), to ligate 3’ and 5’ adapters onto RNA fragments. After each reaction, beads were washed 1x in WBI and 3x in 1xPNK:
(i) Alkaline phosphatase treatment (30 min, 37 °C): 8 U TSAP, 80 U RNasIN.
(ii) 3’ linker ligation (overnight, 16 °C): 0.1 nmol miRCat-33 DNA linker, 40 U T4 RNA Ligase 1, 80 U RNasIN, 12.5 % (v/v) PEG8000.
(iii) 5’ phosphorylation (1 hr, 37 °C): 40 U T4 PNK, 2 ?L ?32P-ATP (after 30 min, add 1 ?L 100 mM rATP and an additional 20 U T4 PNK).
(iv) 5’ linker ligation (overnight, 16 °C): 0.2 nmol 5’ linker, 40 U T4 RNA Ligase 1, 1.25 mM rATP, 80 U RNasIN, 12.5 % (v/v) PEG8000.
After the final reaction, beads were washed 3x in WBII (50 mM Tris-HCl pH 7.8, 50 mM NaCl, 0.1 % Nonidet P40 substitute, 5 mM beta-mercaptoethanol), resuspended in 30 µL 1x NuPAGE LDS sample buffer, heated at 95 °C for 2 min, and the eluate quickly removed and loaded onto a NuPAGE 4-12 % polyacrylamide gel. The gel was run at 100 V for ~1 hr, then protein:RNA complexes transferred to Hybond-C extra nitrocellulose membrane (Amersham) at 150 V for 1.5 hr using a wet transfer system and NuPAGE transfer buffer with 15 % methanol. The membrane was then briefly dried, exposed to BioMax MS film (4 hr to overnight) and the region corresponding to the protein:RNA complex cut out.
The membrane slice was then incubated in 400 ?L WBII with 1 % (w/v) SDS, 5 mM EDTA and 100 ?g Proteinase K at 55 °C for 2 hr. The solution was then removed to another tube, 50 µl 3M NaAc pH 5.2 and 500 µl of 1:1 phenol:chloroform mix added, and the mixture vortexed then centrifuged at 14,000 xg for 20 min. The top phase was transferred into a new tube and 1 ml ethanol and 20 ?g glycogen added. The solution was stored at -20 °C overnight to precipitate RNA, then centrifuged at 14,000 xg for 1 hr. The pellet was washed once with 70 % ethanol and allowed to briefly air dry, before resuspending in 11 ?L water + 1 ?L 10 ?M miRCat-33 RT oligo + 1 ?L 10 mM dNTP mix. The solution was heated to 80 °C for 3 min, snap cooled on ice for 5 min, then the following mix added: 4 ?L 5x first strand buffer (SSIII kit) + 1 ?L 100 mM DTT (SSIII kit) + 1 ?L recombinant RNasIN. After incubating for 3 min at 50 °C, 200 U of SuperScript III was added and the reverse transcription allowed to proceed for 1 hr at 50 °C. The reaction was stopped by heating to 65 °C for 15 min, then RNA digested with 10 U RNase H at 37 °C for 30 min. PCR reactions (80 ?L) were then prepared, each with 2 ?L cDNA, 10 pmol P5, 10 pmol PE, 12.5 nmol each dNTP and 2.5 U LA Taq. Typically, we ran five PCR reactions and then concentrated the products by ethanol precipitation before resolving on a 3 % metaphor agarose gel in 0.5x TBE. A smear corresponding to the size of the two adapters plus inserts (total size ~120-300 bp) was then excised, and DNA extracted using the MinElute gel extraction kit, eluting in 20 ?L water. If the experiment was successful, we repeated the PCRs with the remaining half of the cDNA.
The above CRAC protocol is referred to as the “long” protocol. For some samples, a shorter version of the protocol was used, which did not affect library content. The shorter version omits radiolabelling (using cold rATP instead), SDS-PAGE and transfer to nitrocellulose. Instead, after 3’ linker ligation, beads were washed and added directly to 400 ?L WBII with 1 % (w/v) SDS, 5 mM EDTA and 100 ?g Proteinase K. This version of the CRAC protocol is referred to as the “short” protocol.

tag: tissue

value: mouse embryonic stem cell

tag: genetic background

tag: cell treatment

tag: assay type

value: CRAC (long protocol)

tag: cell lysis conditions

value: RIPA


title: CRAC_rRNA_control_16

description:
in-line barcode (present at 5' end of CRAC reads): NNNNCTAGC
rRNA analysis samples: sample used only for rRNA CRAC analysis


title: E20_MS126_088 (SingleCell579)

description: Cynomolgus Embryo at E20


title: E43f_MS132_213 (SingleCell591)


title: E17_MS127_135 (SingleCell568)

description: Cynomolgus Embryo at E17


title: E17_MS127_134 (SingleCell567)

description: Cynomolgus Embryo at E17


title: E17_MS127_132 (SingleCell565)

description: Cynomolgus Embryo at E17


title: E17_MS126_128 (SingleCell561)

description: Cynomolgus Embryo at E17


title: E17_MS126_127 (SingleCell560)

description: Cynomolgus Embryo at E17


title: E17_MS126_121 (SingleCell554)

description: Cynomolgus Embryo at E17


title: E17_MS127_133 (SingleCell566)

description: Cynomolgus Embryo at E17


title: E17_MS126_122 (SingleCell555)

description: Cynomolgus Embryo at E17


title: E17_MS126_125 (SingleCell558)

description: Cynomolgus Embryo at E17


title: E17_MS127_131 (SingleCell564)

description: Cynomolgus Embryo at E17


title: E17_MS127_130 (SingleCell563)

description: Cynomolgus Embryo at E17


title: E17_MS127_129 (SingleCell562)

description: Cynomolgus Embryo at E17


title: E17_MS126_126 (SingleCell559)

description: Cynomolgus Embryo at E17


title: E17_MS126_124 (SingleCell557)

description: Cynomolgus Embryo at E17


title: E17_MS126_123 (SingleCell556)

description: Cynomolgus Embryo at E17


title: E17_MS127_141 (SingleCell574)

description: Cynomolgus Embryo at E17


title: E17_MS126_120 (SingleCell553)

description: Cynomolgus Embryo at E17


title: E17_MS127_140 (SingleCell573)

description: Cynomolgus Embryo at E17


title: E17_MS126_119 (SingleCell552)

description: Cynomolgus Embryo at E17


title: E17_MS126_118 (SingleCell551)

description: Cynomolgus Embryo at E17


title: E17_MS127_136 (SingleCell569)

description: Cynomolgus Embryo at E17


title: E17_MS127_137 (SingleCell570)

description: Cynomolgus Embryo at E17


title: 47: Ski2Aven.clone7E.d4_rep2_360

data_processing:
Library strategy: RNA half-life analysis
Reads were mapped to mm10, gencode.vM16 annotations, with the following command (for example):
STAR --runMode alignReads --outSAMtype BAM SortedByCoordinate --readFilesIn sample1.fastq --genomeDir /References/gencode.vM16.annotation.withERCCs --runThreadN 12 --outFilterType BySJout --outFilterMultimapNmax 10000 --outFilterMismatchNmax 3 --outSAMmultNmax 1 --outSAMattributes NH HI NM MD AS nM --outMultimapperOrder Random --outSAMunmapped None --quantMode GeneCounts --outFileNamePrefix sample1_
Counts were taken from the "ReadsPerGene.out.tab" tables, and combined for all samples into the table "Counts_for_half_life_analysis.txt".
Half lives were calculated as described previously (PMID: 30456373), except rather than normalising each of the 47 samples internally to ERCCs, samples for each timepoint were first normalised using the "estimateSizeFactorsForMatrix" function from the DESeq2 package in R. Then, median total_mRNA_counts:total_ERCC_counts were calculated for each timepoint (to give a single size factor per timepoint). Samples were then normalised by linear scaling according to this timepoint-specific size factor. This accounts for the fact that total mRNA levels decay across the timecourse (to approximately half of their original level by timepoint 360 minutes).
Half lives are provided in the table "half_lives_231019.txt".
Genome_build: mm10

extract_protocol:
Agilent Absolutely RNA Miniprep Kit
Ribosomal RNA depletion with the Illumina Ribozero kit followed by library preparation with the TruSeq v2 kit, Illumina

value: 5 ug/ul Actinomycin D for 6 hr + 0.1uM 4OHT for 4 days

value: RNA half life measurement 6 hr timepoint


title: 46: Ski2Aven.clone7E.d4_rep1_360


title: 45: Ski2Aven.clone7E.d4_rep3_240

value: 5 ug/ul Actinomycin D for 4 hr + 0.1uM 4OHT for 4 days

value: RNA half life measurement 4 hr timepoint


title: 44: Ski2Aven.clone7E.d4_rep2_240


title: 43: Ski2Aven.clone7E.d4_rep1_240


title: E17_MS126_117 (SingleCell550)

description: Cynomolgus Embryo at E17


title: 48: Ski2Aven.clone7E.d4_rep3_360


title: E17_MS126_116 (SingleCell549)

description: Cynomolgus Embryo at E17


title: E17_MS126_114 (SingleCell547)

description: Cynomolgus Embryo at E17


title: 40: Ski2Aven.clone7E.d4_rep1_120

value: 5 ug/ul Actinomycin D for 2 hr + 0.1uM 4OHT for 4 days

value: RNA half life measurement 2 hr timepoint


title: E17_MS126_112 (SingleCell545)

description: Cynomolgus Embryo at E17


title: 42: Ski2Aven.clone7E.d4_rep3_120


title: E17_MS126_113 (SingleCell546)

description: Cynomolgus Embryo at E17


title: E17_MS126_115 (SingleCell548)

description: Cynomolgus Embryo at E17


title: 41: Ski2Aven.clone7E.d4_rep2_120


title: 38: Ski2Aven.clone7E.d4_rep2_0

value: DMSO 6 hr control + 0.1uM 4OHT for 4 days

value: RNA half life measurement control


title: 39: Ski2Aven.clone7E.d4_rep3_0


title: 36: Ski2Aven.clone7E.d0_rep3_360

value: 5 ug/ul Actinomycin D for 6 hr


title: 37: Ski2Aven.clone7E.d4_rep1_0


title: 35: Ski2Aven.clone7E.d0_rep2_360


title: E17_MS126_107 (SingleCell540)

description: Cynomolgus Embryo at E17


title: E17_MS126_108 (SingleCell541)

description: Cynomolgus Embryo at E17


title: 34: Ski2Aven.clone7E.d0_rep1_360


title: 33: Ski2Aven.clone7E.d0_rep3_240

value: 5 ug/ul Actinomycin D for 4 hr


title: E17_MS126_106 (SingleCell539)

description: Cynomolgus Embryo at E17


title: E17_MS126_105 (SingleCell538)

description: Cynomolgus Embryo at E17


title: E17_MS126_104 (SingleCell537)

description: Cynomolgus Embryo at E17


title: E17_MS126_103 (SingleCell536)

description: Cynomolgus Embryo at E17


title: 31: Ski2Aven.clone7E.d0_rep1_240


title: 32: Ski2Aven.clone7E.d0_rep2_240


title: 30: Ski2Aven.clone7E.d0_rep3_120

value: 5 ug/ul Actinomycin D for 2 hr


title: E17_MS126_102 (SingleCell535)

description: Cynomolgus Embryo at E17

value: not applicable


title: 29: Ski2Aven.clone7E.d0_rep2_120


title: E17_MS126_101 (SingleCell534)

description: Cynomolgus Embryo at E17


title: E17_MS126_111 (SingleCell544)

description: Cynomolgus Embryo at E17


title: 28: Ski2Aven.clone7E.d0_rep1_120


title: E17_MS126_109 (SingleCell542)

description: Cynomolgus Embryo at E17


title: E17_MS126_100 (SingleCell533)

description: Cynomolgus Embryo at E17


title: E17_MS126_098 (SingleCell531)

description: Cynomolgus Embryo at E17


title: 25: Ski2Aven.clone7E.d0_rep1_0

value: DMSO 6 hr control


title: E17_MS126_099 (SingleCell532)

description: Cynomolgus Embryo at E17

value: embryo


title: E17_MS126_097 (SingleCell530)

description: Cynomolgus Embryo at E17


title: 24: Ski2.clone5F.d4_rep3_360

value: mouse embryonic stem cell

value: Rosa26Cre-ERT2/- Skiv2lfl/fl


title: 26: Ski2Aven.clone7E.d0_rep2_0


title: 27: Ski2Aven.clone7E.d0_rep3_0


title: E17_MS126_095 (SingleCell528)

description: Cynomolgus Embryo at E17


title: E17_MS126_094 (SingleCell527)

description: Cynomolgus Embryo at E17


title: 21: Ski2.clone5F.d4_rep3_240


title: E17_MS126_096 (SingleCell529)

description: Cynomolgus Embryo at E17


title: 20: Ski2.clone5F.d4_rep2_240


title: 22: Ski2.clone5F.d4_rep1_360


title: E17_MS126_093 (SingleCell526)

description: Cynomolgus Embryo at E17


title: E17_MS126_092 (SingleCell525)

description: Cynomolgus Embryo at E17


title: E17_MS118_091 (SingleCell523)

description: Cynomolgus Embryo at E17


title: 18: Ski2.clone5F.d4_rep3_120


title: E17_MS126_091 (SingleCell524)

description: Cynomolgus Embryo at E17


title: 17: Ski2.clone5F.d4_rep2_120


title: E17_MS117_091 (SingleCell522)

description: Cynomolgus Embryo at E17


title: 16: Ski2.clone5F.d4_rep1_120


title: E17_MS117_090 (SingleCell521)

description: Cynomolgus Embryo at E17


title: 15: Ski2.clone5F.d4_rep3_0


title: E17_MS117_089 (SingleCell520)

description: Cynomolgus Embryo at E17


title: 23: Ski2.clone5F.d4_rep2_360


title: 14: Ski2.clone5F.d4_rep2_0


title: 13: Ski2.clone5F.d4_rep1_0


title: E16_MS134_071 (SingleCell518)

description: Cynomolgus Embryo at E16

source_name: Cynomolgus Embryo at E16


title: E16_MS134_070 (SingleCell517)

description: Cynomolgus Embryo at E16


title: 10: Ski2.clone5F.d0_rep1_360


title: E16_MS134_069 (SingleCell516)

description: Cynomolgus Embryo at E16


title: 12: Ski2.clone5F.d0_rep3_360


title: 11: Ski2.clone5F.d0_rep2_360


title: 19: Ski2.clone5F.d4_rep1_240


title: E16_MS134_068 (SingleCell515)

description: Cynomolgus Embryo at E16


title: 9: Ski2.clone5F.d0_rep3_240


title: UCC_EU2015_12

value: Upper Tract Urothelial Cell Carcinoma


title: 8: Ski2.clone5F.d0_rep2_240


title: 7: Ski2.clone5F.d0_rep1_240


title: E16_MS134_067 (SingleCell514)

description: Cynomolgus Embryo at E16


title: 6: Ski2.clone5F.d0_rep3_120


title: E16_MS134_063 (SingleCell510)

description: Cynomolgus Embryo at E16


title: E16_MS134_066 (SingleCell513)

description: Cynomolgus Embryo at E16


title: E16_MS134_064 (SingleCell511)

description: Cynomolgus Embryo at E16


title: E16_MS134_062 (SingleCell509)

description: Cynomolgus Embryo at E16


title: 3: Ski2.clone5F.d0_rep3_0


title: 4: Ski2.clone5F.d0_rep1_120


title: 2: Ski2.clone5F.d0_rep2_0


title: E16_MS134_061 (SingleCell508)

description: Cynomolgus Embryo at E16


title: E16_MS133_052 (SingleCell506)

description: Cynomolgus Embryo at E16


title: 1: Ski2.clone5F.d0_rep1_0


title: RNAseq_totalRNA_Aven_KO_rep2

data_processing:
Library strategy: Ribosome profiling input
Ribosome and disome profiling
Preprocessing of Ribosome Footprints
Initial quality assessment of the sequencing reads was conducted based on the preliminary quality values produced by the Illumina pipeline 2.19.1 such as the percentage of clusters passed filtering (%PF clusters) and the mean quality score (PF clusters). Adapter sequences were removed using cutadapt utility (Martin, 2011) with following options: -a AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC --match-read-wildcards. Next, trimmed read sequences were filtered by their size using an in-house Python script with following inclusive ranges: [26,35] for monosome footprints, [45,70] for disome footprints and [21,70] for total RNA. Smaller or larger fragments were kept separately and not used in further analysis. Finally, the reads were filtered for quality using fastq_quality_filter tool from the FASTX-toolkit (http://hannonlab.cshl.edu/fastx toolkit/) with the following arguments: -Q33 -q 30 -p 80.
Mapping of Footprints to Mouse Genome
The preprocessed insert sequences were mapped sequentially to following databases: mouse rRNA, human rRNA, mt-tRNA, mouse tRNA, mouse cDNA from Ensembl mouse database release 91 and, finally, mouse genomic sequences (Genome Reference Consortium GRCm38.p2). In all but the final mapping against genomic sequences, bowtie version 2.3.0 (Langmead and Salzberg, 2012) was used with the following parameters: -p 2 -L 15 -k 20 --no-unal,
After each alignment, only reads that were not aligned were used in the following mapping. For further analysis, only alignments against mouse cDNA were used, unless specifically stated otherwise. For each query sequence, only alignments with maximum alignment scores were kept.
Separately from this sequential alignment strategy, trimmed and filtered total RNA sequences from each sample were also directly aligned against the mouse genome. This mapping and the final mapping of the sequential alignment strategy were performed using STAR version 2.5.3a with the following parameters:
--runThreadN 6 --genomeDir=mouse/star/Mmusculus.GRCm38.91
=--readFilesCommand zcat --genomeLoad LoadAndKeep
=--outSAMtype BAM SortedByCoordinate Unsorted
--alignSJDBoverhangMin 1 --alignIntronMax 1000000
--outFilterType BySJout --alignSJoverhangMin 8
--limitBAMsortRAM 15000000000
The output of this alignment was used to estimate expressed transcript models out of all models contained in Ensembl mouse database release 91. To this end, we used StringTie version 1.3.3b to estimate the number of fragments per kilo base of exon per million fragments mapped (FPKM) for each transcript, with the following parameters:
-p 8 -G Mmusculus.GRCm38.91.gtf -A gene_abund.tab
-C cov_refs.gtf -B -e
The resulting FPKM estimate information was parsed with an in-house Python script to identify transcripts which had an FPKM >0.2 and an isoform abundance fraction >0.05 in at least 2 samples. A database of expressed transcripts based on this filtering was used in further analysis. Among those, genes that were estimated to have a single expressed isoform were annotated as single transcript genes.
Quantification of mRNA and Footprint Abundances and Translational Efficiencies
Abundance of mRNA and monosome or disome protected fragments was estimated per gene as described in (PMID: 26486724). For this quantification, only reads that were mapped uniquely to a single gene and only to transcripts that were identified to be expressed (see Mapping of Footprints to Mouse Genome) were used. We used a limited size range of disome fragments (56-64 nt), as this facilitated subsequent A-site assignment and high resolution analysis of stall sites.
Read counts of total RNA and RPF were normalized with upper quantile method of R package edgeR v3.16.5. Prior to normalization, transcripts which did not have at least 10 counts in at least one third of the samples were removed from the datasets. For better comparability between datasets, RPKM values were calculated as the number of counted reads per 1000 mappable and countable bases per geometric mean of normalized read counts per million. Genes that had an average total RNA RPKM >5 were designated as robustly expressed.
Translational efficiencies (TE) were then calculated as the ratio of footprint-RPKM to total RNA-RPKM for mososomes and disomes per sample. For most analysis downstream, TEs were log2 transformed. Significant changes in abundances of total RNA, monosomes and disomes between control and treated samples were assessed by DESeq2 package for the R statistical environment. Significant changes in TEs of monosomes and disomes between control and treated samples were assessed by R package xtail. The false discovery rate (FDR) adjusted p-values were used to identify statistically significant changes at 0.05 FDR.
Analysis of Monosome and Disome Positions on Transcripts
For total RNA and RPF reads that were counted towards genes, we have also tracked the position of the reads relative to the 5’ end of its corresponding transcript. For total RNA reads and monosome footprints we have used the 5’ end of the reads and the estimated A-site of the ribosomes, respectively, as described in (PMID: 26486724). For disome footprints we have established an empirical offsetting scheme based on the size and the frame (relative to the main CDS) of the footprints. We estimated the A-site of the pausing ribosome at the disome site by adding 45, 44, or 43 to the map position of the 5’ end of 58nt-long foot- prints that were at 1st, 2nd or 3rd frame, respectively. Similarly, we used the following offsets for 59nt, 60nt, 62nt and 63nt long disome footprints, respectively: [45, 44, 46], [45, 44, 46], [48, 47, 46], [48, 47, 49]. These coordinates then were converted into Wiggle Track Format (WIG) by in-house Python scripts.
processed data files format and content: For CRAC, bigwig files are provided. Values are reads per million, and replicates for the same protein are pooled.
processed data files format and content: For RNA-seq, differential expression was analysed using DESeq2 and values are provided in Supplementary_table_3.txt. This table also contains some processed CRAC counts, that were used in conjunction with the RNA-seq analysis to elucidate the functions of SKIV2L and AVEN. Supplementary_table_3.txt contains the following columns:
processed data files format and content: Column 1: Gene ID.
processed data files format and content: Columns 2-3: Half-life analysis from PMID: 30456373.
processed data files format and content: Columns 4-7: Relative CRAC counts for Aven, Mtr4, Xrn1 and Skiv2l. For each gene, the APPRIS principal isoform is used for counting (picking one at random in case there are multiple).
processed data files format and content: Columns 8-9: tSNE coordinates based on relative CRAC counts for Mtr4, Xrn1 and Skiv2l, used to arrange transcripts by their relative binding to these three factors (which reflect RNA degradation by the three major mammalian decay pathways).
processed data files format and content: Column 10: Transcript ID used for CRAC counting.
processed data files format and content: Column 11: Gene name.
processed data files format and content: Columns 12-14: CDS and transcript length details.
processed data files format and content: Columns 15-18: RNA-seq RPKM values for Mtr4, Xrn1 and Skiv2l cell lines used for CRAC (3xFLAG-Avi-tagged lines).
processed data files format and content: Columns 19-21: Relative CRAC counts for Mtr4, Xrn1 and Skiv2l normalized to RNA-seq.
processed data files format and content: Columns 22-30: DESeq2 base mean, log-fold change and adjusted p value for Skiv2lfl/fl knock out after 2, 4 or 6 days of 4OHT addition, versus untreated.
processed data files format and content: Columns 31-33: DESeq2 base mean, log-fold change and adjusted p value for Aven-/- versus wild-type mESCs.
processed data files format and content: Columns 34-39: DESeq2 base mean, log-fold change and adju

extract_protocol: Cells were harvested (without cycloheximide pretreatment) and flash-frozen in liquid nitrogen. RNA-seq libraries were prepared essentially following the Illumina protocol:  after total RNA extraction using miRNeasy RNA Extraction kit (Qiagen), ribosomal RNA was depleted using Ribo-Zero Gold rRNA (Illumina), and sequencing libraries generated from the heat-fragmented RNA.

value: Ribosome profiling input


title: E16_MS133_051 (SingleCell505)

description: Cynomolgus Embryo at E16


title: E16_MS133_050 (SingleCell504)

description: Cynomolgus Embryo at E16


title: E16_MS133_049 (SingleCell503)

description: Cynomolgus Embryo at E16


title: RNAseq_totalRNA_WT_rep1


title: E16_MS133_048 (SingleCell502)

description: Cynomolgus Embryo at E16


title: RNAseq_totalRNA_Aven_KO_rep1


title: E16_MS133_047 (SingleCell501)

description: Cynomolgus Embryo at E16


title: E16_MS132_241 (SingleCell499)

description: Cynomolgus Embryo at E16


title: E16_MS132_235 (SingleCell498)

description: Cynomolgus Embryo at E16


title: RiboSeq_ribosome_Aven_KO_rep1

data_processing:
Library strategy: Ribosome profiling
Ribosome and disome profiling
Preprocessing of Ribosome Footprints
Initial quality assessment of the sequencing reads was conducted based on the preliminary quality values produced by the Illumina pipeline 2.19.1 such as the percentage of clusters passed filtering (%PF clusters) and the mean quality score (PF clusters). Adapter sequences were removed using cutadapt utility (Martin, 2011) with following options: -a AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC --match-read-wildcards. Next, trimmed read sequences were filtered by their size using an in-house Python script with following inclusive ranges: [26,35] for monosome footprints, [45,70] for disome footprints and [21,70] for total RNA. Smaller or larger fragments were kept separately and not used in further analysis. Finally, the reads were filtered for quality using fastq_quality_filter tool from the FASTX-toolkit (http://hannonlab.cshl.edu/fastx toolkit/) with the following arguments: -Q33 -q 30 -p 80.
Mapping of Footprints to Mouse Genome
The preprocessed insert sequences were mapped sequentially to following databases: mouse rRNA, human rRNA, mt-tRNA, mouse tRNA, mouse cDNA from Ensembl mouse database release 91 and, finally, mouse genomic sequences (Genome Reference Consortium GRCm38.p2). In all but the final mapping against genomic sequences, bowtie version 2.3.0 (Langmead and Salzberg, 2012) was used with the following parameters: -p 2 -L 15 -k 20 --no-unal,
After each alignment, only reads that were not aligned were used in the following mapping. For further analysis, only alignments against mouse cDNA were used, unless specifically stated otherwise. For each query sequence, only alignments with maximum alignment scores were kept.
Separately from this sequential alignment strategy, trimmed and filtered total RNA sequences from each sample were also directly aligned against the mouse genome. This mapping and the final mapping of the sequential alignment strategy were performed using STAR version 2.5.3a with the following parameters:
--runThreadN 6 --genomeDir=mouse/star/Mmusculus.GRCm38.91
=--readFilesCommand zcat --genomeLoad LoadAndKeep
=--outSAMtype BAM SortedByCoordinate Unsorted
--alignSJDBoverhangMin 1 --alignIntronMax 1000000
--outFilterType BySJout --alignSJoverhangMin 8
--limitBAMsortRAM 15000000000
The output of this alignment was used to estimate expressed transcript models out of all models contained in Ensembl mouse database release 91. To this end, we used StringTie version 1.3.3b to estimate the number of fragments per kilo base of exon per million fragments mapped (FPKM) for each transcript, with the following parameters:
-p 8 -G Mmusculus.GRCm38.91.gtf -A gene_abund.tab
-C cov_refs.gtf -B -e
The resulting FPKM estimate information was parsed with an in-house Python script to identify transcripts which had an FPKM >0.2 and an isoform abundance fraction >0.05 in at least 2 samples. A database of expressed transcripts based on this filtering was used in further analysis. Among those, genes that were estimated to have a single expressed isoform were annotated as single transcript genes.
Quantification of mRNA and Footprint Abundances and Translational Efficiencies
Abundance of mRNA and monosome or disome protected fragments was estimated per gene as described in (PMID: 26486724). For this quantification, only reads that were mapped uniquely to a single gene and only to transcripts that were identified to be expressed (see Mapping of Footprints to Mouse Genome) were used. We used a limited size range of disome fragments (56-64 nt), as this facilitated subsequent A-site assignment and high resolution analysis of stall sites.
Read counts of total RNA and RPF were normalized with upper quantile method of R package edgeR v3.16.5. Prior to normalization, transcripts which did not have at least 10 counts in at least one third of the samples were removed from the datasets. For better comparability between datasets, RPKM values were calculated as the number of counted reads per 1000 mappable and countable bases per geometric mean of normalized read counts per million. Genes that had an average total RNA RPKM >5 were designated as robustly expressed.
Translational efficiencies (TE) were then calculated as the ratio of footprint-RPKM to total RNA-RPKM for mososomes and disomes per sample. For most analysis downstream, TEs were log2 transformed. Significant changes in abundances of total RNA, monosomes and disomes between control and treated samples were assessed by DESeq2 package for the R statistical environment. Significant changes in TEs of monosomes and disomes between control and treated samples were assessed by R package xtail. The false discovery rate (FDR) adjusted p-values were used to identify statistically significant changes at 0.05 FDR.
Analysis of Monosome and Disome Positions on Transcripts
For total RNA and RPF reads that were counted towards genes, we have also tracked the position of the reads relative to the 5’ end of its corresponding transcript. For total RNA reads and monosome footprints we have used the 5’ end of the reads and the estimated A-site of the ribosomes, respectively, as described in (PMID: 26486724). For disome footprints we have established an empirical offsetting scheme based on the size and the frame (relative to the main CDS) of the footprints. We estimated the A-site of the pausing ribosome at the disome site by adding 45, 44, or 43 to the map position of the 5’ end of 58nt-long foot- prints that were at 1st, 2nd or 3rd frame, respectively. Similarly, we used the following offsets for 59nt, 60nt, 62nt and 63nt long disome footprints, respectively: [45, 44, 46], [45, 44, 46], [48, 47, 46], [48, 47, 49]. These coordinates then were converted into Wiggle Track Format (WIG) by in-house Python scripts.
processed data files format and content: For CRAC, bigwig files are provided. Values are reads per million, and replicates for the same protein are pooled.
processed data files format and content: For RNA-seq, differential expression was analysed using DESeq2 and values are provided in Supplementary_table_3.txt. This table also contains some processed CRAC counts, that were used in conjunction with the RNA-seq analysis to elucidate the functions of SKIV2L and AVEN. Supplementary_table_3.txt contains the following columns:
processed data files format and content: Column 1: Gene ID.
processed data files format and content: Columns 2-3: Half-life analysis from PMID: 30456373.
processed data files format and content: Columns 4-7: Relative CRAC counts for Aven, Mtr4, Xrn1 and Skiv2l. For each gene, the APPRIS principal isoform is used for counting (picking one at random in case there are multiple).
processed data files format and content: Columns 8-9: tSNE coordinates based on relative CRAC counts for Mtr4, Xrn1 and Skiv2l, used to arrange transcripts by their relative binding to these three factors (which reflect RNA degradation by the three major mammalian decay pathways).
processed data files format and content: Column 10: Transcript ID used for CRAC counting.
processed data files format and content: Column 11: Gene name.
processed data files format and content: Columns 12-14: CDS and transcript length details.
processed data files format and content: Columns 15-18: RNA-seq RPKM values for Mtr4, Xrn1 and Skiv2l cell lines used for CRAC (3xFLAG-Avi-tagged lines).
processed data files format and content: Columns 19-21: Relative CRAC counts for Mtr4, Xrn1 and Skiv2l normalized to RNA-seq.
processed data files format and content: Columns 22-30: DESeq2 base mean, log-fold change and adjusted p value for Skiv2lfl/fl knock out after 2, 4 or 6 days of 4OHT addition, versus untreated.
processed data files format and content: Columns 31-33: DESeq2 base mean, log-fold change and adjusted p value for Aven-/- versus wild-type mESCs.
processed data files format and content: Columns 34-39: DESeq2 base mean, log-fold change and adjusted p

extract_protocol: Cells were harvested (without cycloheximide pretreatment) and flash-frozen in liquid nitrogen. From the cell pellets, lysates were prepared and ribosome-protected mRNA fragments were generated by RNase I. For the excision of footprints from 15% urea-polyacrylamide gels, single strand RNA oligonucleotides of 26 nt and 34 nt were used as size markers for excision of ribosome footprints. After fragment purification with miRNeasy RNA Extraction kit (217004 Qiagen), 5µg fragmented RNA was used for ribosomal RNA removal using Ribo-Zero Gold rRNA Removal Kit (MRZG12324 Illumina). Sequencing libraries were generated according to Illumina’s TruSeq Ribo-Profile (RPHMR12126 Illumina) protocol with minor modifications. cDNA fragments were separated on a 10% urea-polyacrylamide gel and gel slices between 70-80 nt. The PCR-amplified libraries were size selected on an 8% native polyacrylamide gel. Ribosome (monosome) libraries were at ~150 bp.

value: Ribosome profiling


title: RiboSeq_ribosome_WT_rep1