title: RiboSeq_disome_Aven_KO_rep2

data_processing:
Library strategy: Disome 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 v

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 52 nt and 69 nt were used as size markers for excision of disome 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 97-114 nt. The PCR-amplified libraries were size selected on an 8% native polyacrylamide gel. Disome libraries were at ~180 bp.

value: Disome profiling


title: RiboSeq_disome_WT_rep2

status: Public on Jan 29 2020

type: SRA

city: Basel

first: Alex

middle: Charles

last: Tuck

address: Maulbeerstrasse 66

country: Switzerland

institute: Friedrich Miescher Institute for Biomedical Research

library_source: transcriptomic

molecule: total RNA

organism: Mus musculus

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)

tag: tissue

value: mouse embryonic stem cell

tag: genetic background

tag: assay type


title: RiboSeq_disome_Aven_KO_rep1


title: E16_MS134_065 (SingleCell512)

city: Kyoto

first: Yukihiro

last: Yabuta

state: Kyoto

address: Yoshida-Konoe-cho, Sakyo-ku

department: Anatomy and Cell Biology

country: Japan

institute: Kyoto University, Graduate school of medicine

description: Cynomolgus Embryo at E16

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: Cynomolgus Embryo at E16

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.

tag: cell line

value: not applicable

tag: cell type

value: embryo

tag: developmental stage


title: RiboSeq_ribosome_Aven_KO_rep2

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: E16_MS132_230 (SingleCell496)

description: Cynomolgus Embryo at E16


title: CRAC_rRNA_control_47

description:
in-line barcode (present at 5' end of CRAC reads): NNNNGACTTAGC
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

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: cell treatment

value: CRAC (long protocol)

tag: cell lysis conditions

value: RIPA


title: E16_MS134_060 (SingleCell507)

description: Cynomolgus Embryo at E16


title: E16_MS132_231 (SingleCell497)

description: Cynomolgus Embryo at E16


title: E16_MS132_228 (SingleCell495)

description: Cynomolgus Embryo at E16


title: RNAseq_totalRNA_WT_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_MS132_227 (SingleCell494)

description: Cynomolgus Embryo at E16


title: CRAC_rRNA_control_34

data_processing:
Library strategy: CRAC (short 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

value: CRAC (short protocol)


title: RiboSeq_disome_WT_rep1


title: CRAC_Aven_4


title: CRAC_rRNA_control_42


title: E16_MS132_226 (SingleCell493)

description: Cynomolgus Embryo at E16


title: E16_MS132_223 (SingleCell491)

description: Cynomolgus Embryo at E16


title: CRAC_rRNA_control_43

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


title: E16_MS132_224 (SingleCell492)

description: Cynomolgus Embryo at E16


title: RiboSeq_ribosome_WT_rep2


title: CRAC_rRNA_control_30


title: E16_MS132_221 (SingleCell490)

description: Cynomolgus Embryo at E16


title: E16_MS134_072 (SingleCell519)

description: Cynomolgus Embryo at E16


title: E16_MS132_218 (SingleCell488)

description: Cynomolgus Embryo at E16


title: E16_MS132_220 (SingleCell489)

description: Cynomolgus Embryo at E16


title: CRAC_rRNA_control_14


title: E16_MS132_124 (SingleCell486)

description: Cynomolgus Embryo at E16


title: CRAC_rRNA_control_27


title: E16_MS132_116 (SingleCell485)

description: Cynomolgus Embryo at E16


title: CRAC_rRNA_control_8

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


title: E16_MS132_115 (SingleCell484)

description: Cynomolgus Embryo at E16


title: E16_MS132_105 (SingleCell483)

description: Cynomolgus Embryo at E16


title: E17_MS126_110 (SingleCell543)

description: Cynomolgus Embryo at E17

source_name: Cynomolgus Embryo at E17


title: CRAC_rRNA_control_29


title: CRAC_rRNA_control_45


title: CRAC_rRNA_control_38


title: E16_MS132_099 (SingleCell482)

description: Cynomolgus Embryo at E16


title: CRAC_rRNA_control_35


title: E16_MS132_098 (SingleCell481)

description: Cynomolgus Embryo at E16


title: E16_MS132_090 (SingleCell480)

description: Cynomolgus Embryo at E16


title: CRAC_Skiv2l_7


title: E16_MS132_087 (SingleCell479)

description: Cynomolgus Embryo at E16


title: E16_MS132_085 (SingleCell478)

description: Cynomolgus Embryo at E16


title: CRAC_Skiv2l_8


title: E16_MS132_073 (SingleCell477)

description: Cynomolgus Embryo at E16


title: E16_MS132_072 (SingleCell476)

description: Cynomolgus Embryo at E16


title: CRAC_rRNA_control_49


title: E16_MS127_165 (SingleCell475)

description: Cynomolgus Embryo at E16


title: CRAC_rRNA_control_13

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


title: CRAC_rRNA_control_12


title: E16_MS127_163 (SingleCell473)

description: Cynomolgus Embryo at E16


title: CRAC_rRNA_control_41


title: E16_MS127_164 (SingleCell474)

description: Cynomolgus Embryo at E16


title: E16_MS132_201 (SingleCell487)

description: Cynomolgus Embryo at E16


title: E16_MS127_161 (SingleCell472)

description: Cynomolgus Embryo at E16


title: CRAC_rRNA_control_40

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

value: no crosslinking


title: CRAC_rRNA_control_1


title: CRAC_Skiv2l_9


title: CRAC_rRNA_control_6


title: E16_MS126_085 (SingleCell467)

description: Cynomolgus Embryo at E16


title: CRAC_rRNA_control_10

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


title: CRAC_Skiv2l_10


title: E16_MS126_084 (SingleCell466)

description: Cynomolgus Embryo at E16


title: CRAC_rRNA_control_21


title: E16_MS127_157 (SingleCell470)

description: Cynomolgus Embryo at E16


title: CRAC_rRNA_control_48


title: CRAC_rRNA_control_44_lane2


title: E16_MS126_090 (SingleCell469)

description: Cynomolgus Embryo at E16


title: CRAC_rRNA_control_28


title: CRAC_rRNA_control_44_lane1


title: CRAC_rRNA_control_51

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


title: E16_MS127_159 (SingleCell471)

description: Cynomolgus Embryo at E16


title: E16_MS126_081 (SingleCell463)

description: Cynomolgus Embryo at E16


title: E16_MS126_080 (SingleCell462)

description: Cynomolgus Embryo at E16


title: CRAC_Aven_noXL


title: E16_MS126_078 (SingleCell460)

description: Cynomolgus Embryo at E16


title: CRAC_rRNA_control_22


title: E16_MS126_077 (SingleCell459)

description: Cynomolgus Embryo at E16


title: E16_MS126_079 (SingleCell461)

description: Cynomolgus Embryo at E16


title: CRAC_Xrn1_7

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


title: CRAC_rRNA_control_33


title: E16_MS126_086 (SingleCell468)

description: Cynomolgus Embryo at E16


title: CRAC_rRNA_control_46


title: CRAC_rRNA_control_5


title: E16_MS118_156 (SingleCell456)

description: Cynomolgus Embryo at E16


title: E16_MS126_083 (SingleCell465)

description: Cynomolgus Embryo at E16


title: E16_MS118_155 (SingleCell455)

description: Cynomolgus Embryo at E16


title: E16_MS118_152 (SingleCell452)

description: Cynomolgus Embryo at E16


title: E16_MS118_154 (SingleCell454)

description: Cynomolgus Embryo at E16


title: CRAC_Aven_5


title: CRAC_rRNA_control_50

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


title: E16_MS126_082 (SingleCell464)

description: Cynomolgus Embryo at E16


title: E16_MS118_151 (SingleCell451)

description: Cynomolgus Embryo at E16


title: CRAC_rRNA_control_25


title: CRAC_rRNA_control_3


title: E16_MS126_076 (SingleCell458)

description: Cynomolgus Embryo at E16


title: E16_MS118_013 (SingleCell450)

description: Cynomolgus Embryo at E16


title: E16_MS118_012 (SingleCell449)

description: Cynomolgus Embryo at E16


title: CRAC_rRNA_control_26

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


title: E16_MS118_157 (SingleCell457)

description: Cynomolgus Embryo at E16


title: CRAC_rRNA_control_36


title: E16_MS118_011 (SingleCell448)

description: Cynomolgus Embryo at E16


title: CRAC_rRNA_control_18


title: E16_MS118_153 (SingleCell453)

description: Cynomolgus Embryo at E16


title: CRAC_Xrn1_6


title: E16_MS118_010 (SingleCell447)

description: Cynomolgus Embryo at E16


title: CRAC_Skiv2l_11


title: CRAC_rRNA_control_23


title: CRAC_rRNA_control_32


title: E16_MS118_008 (SingleCell445)

description: Cynomolgus Embryo at E16


title: E16_MS118_007 (SingleCell444)

description: Cynomolgus Embryo at E16


title: E16_MS118_005 (SingleCell442)

description: Cynomolgus Embryo at E16


title: E16_MS118_006 (SingleCell443)

description: Cynomolgus Embryo at E16


title: E16_MS118_004 (SingleCell441)

description: Cynomolgus Embryo at E16


title: E16_MS118_003 (SingleCell440)

description: Cynomolgus Embryo at E16


title: E16_MS118_002 (SingleCell439)

description: Cynomolgus Embryo at E16


title: CRAC_rRNA_control_17


title: CRAC_rRNA_control_20


title: CRAC_rRNA_control_24

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


title: CRAC_rRNA_control_4


title: E16_MS117_088 (SingleCell437)

description: Cynomolgus Embryo at E16


title: CRAC_rRNA_control_31


title: E16_MS117_086 (SingleCell436)

description: Cynomolgus Embryo at E16


title: E16_MS117_111 (SingleCell438)

description: Cynomolgus Embryo at E16


title: CRAC_rRNA_control_7


title: CRAC_rRNA_control_52

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


title: CRAC_rRNA_control_19


title: E16_MS117_085 (SingleCell435)

description: Cynomolgus Embryo at E16


title: CRAC_rRNA_control_9


title: E16_MS118_009 (SingleCell446)

description: Cynomolgus Embryo at E16


title: RNAseq_Skiv2l_conditional_KO_9E_d6_rep1

data_processing:
RNA-seq: RNA-seq reads were aligned to mm10 and counted using STAR_2.5.0a (--runMode alignReads –outSAMtype BAM SortedByCoordinate --outFilterType BySJout --outFilterMultimapNmax 1 --outFilterMismatchNmax 3 --outSAMmultNmax 1 --outSAMattributes NH HI NM MD AS nM --outMultimapperOrder Random --outSAMunmapped None --quantMode GeneCounts). The STAR index was made using the Mus_musculus.GRCm38.75.dna.primary_assembly.fa file, providing gencode.vM16.annotation.gtf as the sjdbGTF file. DESeq2 was used to test for differential gene expression, with the model formula including biological clone (where at least two were available, see title), sequencing batch (where more than one was performed) and genotype/treatment (WT versus knockout, or time of 4OHT treatment).
For CRAC, bigwig files are provided. Values are reads per million, and replicates for the same protein are pooled.
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:
Column 1: Gene ID.
Columns 2-3: Half-life analysis from PMID: 30456373.
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).
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).
Column 10: Transcript ID used for CRAC counting.
Column 11: Gene name.
Columns 12-14: CDS and transcript length details.
Columns 15-18: RNA-seq RPKM values for Mtr4, Xrn1 and Skiv2l cell lines used for CRAC (3xFLAG-Avi-tagged lines).
Columns 19-21: Relative CRAC counts for Mtr4, Xrn1 and Skiv2l normalized to RNA-seq.
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.
Columns 31-33: DESeq2 base mean, log-fold change and adjusted p value for Aven-/- versus wild-type mESCs.
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

extract_protocol: Ribosomal RNA depletion with the Illumina Ribozero kit followed by library preparation with the ScriptSeq v2 kit, Epicentre

value: Rosa26Cre-ERT2/- Skiv2lfl/fl

value: 0.1uM 4OHT for 6 days

value: RNA-seq (batch 2)

value: Agilent Absolutely RNA Miniprep Kit


title: E16_MS107_107 (SingleCell433)

description: Cynomolgus Embryo at E16


title: RNAseq_Skiv2l_conditional_KO_9E_d4_rep1

value: 0.1uM 4OHT for 4 days


title: RNAseq_Skiv2l_conditional_KO_9E_d2_rep1

value: 0.1uM 4OHT for 2 days


title: CRAC_rRNA_control_39


title: E16_MS107_106 (SingleCell432)

description: Cynomolgus Embryo at E16


title: CRAC_rRNA_control_11


title: CRAC_rRNA_control_37


title: E16_MS107_104 (SingleCell431)

description: Cynomolgus Embryo at E16


title: E16_MS107_103 (SingleCell430)

description: Cynomolgus Embryo at E16


title: E16_MS107_102 (SingleCell429)

description: Cynomolgus Embryo at E16


title: E16_MS107_101 (SingleCell428)

description: Cynomolgus Embryo at E16


title: RNAseq_Skiv2l_conditional_KO_9E_d0_rep1


title: RNAseq_Skiv2l_conditional_KO_5F_d6_rep1


title: RNAseq_Skiv2l_conditional_KO_5F_d2_rep1


title: RNAseq_Skiv2l_conditional_KO_5F_d4_rep1


title: E16_MS107_100 (SingleCell427)

description: Cynomolgus Embryo at E16


title: E16_MS107_097 (SingleCell425)

description: Cynomolgus Embryo at E16


title: E16_MS107_096 (SingleCell424)

description: Cynomolgus Embryo at E16


title: RNAseq_ 3xFLAG-Avi-Xrn1

value: RNA-seq (batch 1)


title: E16_MS107_094 (SingleCell422)

description: Cynomolgus Embryo at E16


title: RNAseq_ 3xFLAG-Avi-Skiv2l_deltaAven_6G_rep1


title: RNAseq_Skiv2l_conditional_KO_5F_d0_rep1


title: RNAseq_ 3xFLAG-Avi-Skiv2l_rep1


title: E16_MS107_095 (SingleCell423)

description: Cynomolgus Embryo at E16


title: E16_MS117_082 (SingleCell434)

description: Cynomolgus Embryo at E16


title: RNAseq_ 3xFLAG-Avi-Mtr4


title: RNAseq_ 3xFLAG-Avi-Skiv2l_deltaAven_4H_rep1


title: RNAseq_ 3xFLAG-Avi-Skiv2l_rep2


title: RNAseq_Skiv2l_conditional_KO_Aven_KO_12E_d4

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

value: RNA-seq (batch 3)


title: E16_MS107_086 (SingleCell418)

description: Cynomolgus Embryo at E16


title: E16_MS107_089 (SingleCell420)

description: Cynomolgus Embryo at E16


title: E16_MS107_088 (SingleCell419)

description: Cynomolgus Embryo at E16


title: RNAseq_Skiv2l_conditional_KO_Aven_KO_12E_d2

value: mouse embryonic stem cell


title: E16_MS107_084 (SingleCell417)

description: Cynomolgus Embryo at E16

value: not applicable


title: RNAseq_Skiv2l_conditional_KO_Aven_KO_12E_d0


title: E16_MS107_083 (SingleCell416)

description: Cynomolgus Embryo at E16

value: embryo


title: RNAseq_Skiv2l_conditional_KO_Aven_KO_7E_d2


title: RNAseq_Skiv2l_conditional_KO_Aven_KO_7E_d4


title: E16_MS107_099 (SingleCell426)

description: Cynomolgus Embryo at E16


title: E16_MS107_082 (SingleCell415)

description: Cynomolgus Embryo at E16


title: E16_MS107_081 (SingleCell414)

description: Cynomolgus Embryo at E16


title: E16_MS107_093 (SingleCell421)

description: Cynomolgus Embryo at E16


title: RNAseq_Skiv2l_conditional_KO_9E_d2_rep2


title: RNAseq_Skiv2l_conditional_KO_9E_d4_rep2


title: E16_MS107_080 (SingleCell413)

description: Cynomolgus Embryo at E16


title: E16_MS107_079 (SingleCell412)

description: Cynomolgus Embryo at E16


title: RNAseq_Skiv2l_conditional_KO_9E_d0_rep2


title: E16_MS107_078 (SingleCell411)

description: Cynomolgus Embryo at E16


title: RNAseq_Skiv2l_conditional_KO_5F_d2_rep2


title: RNAseq_ 3xFLAG-Avi-Skiv2l_deltaAven_6G_rep2


title: E16_MS107_077 (SingleCell410)

description: Cynomolgus Embryo at E16


title: E16_MS102_018 (SingleCell408)

description: Cynomolgus Embryo at E16


title: RNAseq_Skiv2l_conditional_KO_5F_d4_rep2


title: RNAseq_Skiv2l_conditional_KO_5F_d0_rep2


title: E16_MS102_019 (SingleCell409)

description: Cynomolgus Embryo at E16


title: E16_MS102_017 (SingleCell407)

description: Cynomolgus Embryo at E16


title: RNAseq_Skiv2l_conditional_KO_Aven_KO_7E_d0


title: E16_MS102_016 (SingleCell406)

description: Cynomolgus Embryo at E16


title: RNAseq_ 3xFLAG-Avi-Skiv2l_deltaAven_4H_rep2


title: E16_MS101_139 (SingleCell404)

description: Cynomolgus Embryo at E16


title: CRAC_Xrn1_5

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

data_processing:
Library strategy: CRAC
CRAC: CRAC reads were preprocessed with the FASTX Toolkit 0.0.14. 3’ adapters (TGGAATTCTCGGGTGCCAAGGC) were removed with fastx_clipper, low quality bases trimmed/reads removed using fastq_quality_trimmer -t 25 and fastq_quality_filter -q 20 -p 90, and sequencing artefacts removed using fastx_artifacts_filter. Duplicate reads (including UMI) were collapsed, then pyCRAC used to split samples by their inline barcodes and extract the UMIs . Low complexity regions were removed (“low complexity stripping”) from the 3’ end of sequences (defined as stretches of 2 nt more where 80 % of positions are the same nucleotide, e.g. AAAAGAA), and prinseq-lite-0.20.4 used as an additional filter to remove low complexity reads (settings -lc_threshold 20 -lc_method dust). We then applied a set of filters to obtain uniquely mapping reads, and remove reads mapping to repeats or abundant non-coding RNAs (e.g. tRNA, snoRNA or rRNA). For this, reads were separately mapped with bowtie2-2.3.4.1 (settings --local -p 10 -a --very-sensitive) to three indexes:
** Genome: mm10 genomic sequence.
** Gencode non-coding RNAs: Gencode release M16 Mt_rRNA, Mt_tRNA, miRNA, rRNA, scRNA, snoRNA, sRNA, scaRNA and snRNA features from the file gencode.vM16.annotation.gtf, and predicted tRNAs from the file gencode.vM16.tRNAs.gtf, and a repeat-masked version of mouse pre-rRNA.
** Gencode mRNAs/lincRNAs: Gencode release M16 protein_coding and lincRNA features from the file gencode.vM16.annotation.gtf.
Reads were assigned as “repeats/ncRNAs” and excluded if they mapped best or equally well either to regions of the genome overlapping RepeatMasker repeats (downloaded from UCSC table browser, version 2012-02-07) or to Gencode non-coding RNAs. From the remaining reads, those with a genome score better/equal to the mRNA/lincRNA score were selected. Further filters were applied:
** Genome alignment is not 5' soft clipped
** MAPQ >= 8, and second best alignment < 0.8 * best alignment score (or NA)
** Collapse by genomic 5' end position (including strand) and barcode
Replicates for the same protein were then pooled and normalised to counts per million, and converted into bigWig filtes.
For CRAC, bigwig files are provided. Values are reads per million, and replicates for the same protein are pooled.
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:
Column 1: Gene ID.
Columns 2-3: Half-life analysis from PMID: 30456373.
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).
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).
Column 10: Transcript ID used for CRAC counting.
Column 11: Gene name.
Columns 12-14: CDS and transcript length details.
Columns 15-18: RNA-seq RPKM values for Mtr4, Xrn1 and Skiv2l cell lines used for CRAC (3xFLAG-Avi-tagged lines).
Columns 19-21: Relative CRAC counts for Mtr4, Xrn1 and Skiv2l normalized to RNA-seq.
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.
Columns 31-33: DESeq2 base mean, log-fold change and adjusted p value for Aven-/- versus wild-type mESCs.
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

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 μL TN150 supplemented with 5 mM beta-mercaptoethanol and 1 μL of 0.1 U diluted RNace-IT. After 4 min at 37 °C, the RNases were denatured by replacing the solution with 400 μL 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 μL 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 μL 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 μL γ32P-ATP (after 30 min, add 1 μL 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 μL WBII with 1 % (w/v) SDS, 5 mM EDTA and 100 μg 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 μg 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 μL water + 1 μL 10 μM miRCat-33 RT oligo + 1 μL 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 μL 5x first strand buffer (SSIII kit) + 1 μL 100 mM DTT (SSIII kit) + 1 μL 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 μL) were then prepared, each with 2 μL 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 μL water. If the experiment was successful, we repeated the PCRs with the remaining half of the cDNA.
The 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 μL WBII with 1 % (w/v) SDS, 5 mM EDTA and 100 μg Proteinase K. This version of the CRAC protocol is referred to as the “short” protocol.


title: E16_MS101_138 (SingleCell403)

description: Cynomolgus Embryo at E16


title: E16_MS101_137 (SingleCell402)

description: Cynomolgus Embryo at E16


title: CRAC_Xrn1_3


title: CRAC_Xrn1_4

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


title: CRAC_Xrn1_2

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


title: E16_MS101_133 (SingleCell399)

description: Cynomolgus Embryo at E16


title: CRAC_Skiv2l_Focad_KO_4B_1

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


title: CRAC_Skiv2l_Focad_KO_2F_1

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


title: E16_MS101_132 (SingleCell398)

description: Cynomolgus Embryo at E16


title: E16_MS101_134 (SingleCell400)

description: Cynomolgus Embryo at E16


title: RNAseq_ 3xFLAG-Avi-Skiv2l_rep4


title: E16_MS101_140 (SingleCell405)

description: Cynomolgus Embryo at E16


title: RNAseq_ 3xFLAG-Avi-Skiv2l_rep3

value: RNA-seq


title: E16_MS101_130 (SingleCell396)

description: Cynomolgus Embryo at E16


title: E16_MS101_131 (SingleCell397)

description: Cynomolgus Embryo at E16


title: CRAC_Skiv2l_Aven_KO_6G_2


title: CRAC_Skiv2l_Aven_KO_6G_1

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


title: CRAC_Skiv2l_Aven_KO_6G_3

value: CRAC (long protocol)


title: CRAC_Skiv2l_Aven_KO_4F_2

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


title: E16_MS101_126 (SingleCell393)

description: Cynomolgus Embryo at E16


title: E16_MS101_127 (SingleCell394)

description: Cynomolgus Embryo at E16


title: E16_MS101_135 (SingleCell401)

description: Cynomolgus Embryo at E16


title: E16_MS101_125 (SingleCell392)

description: Cynomolgus Embryo at E16


title: E16_MS101_123 (SingleCell391)

description: Cynomolgus Embryo at E16


title: CRAC_Skiv2l_Aven_KO_4F_1

value: RIPA


title: E16_MS101_122 (SingleCell390)

description: Cynomolgus Embryo at E16


title: CRAC_Skiv2l_Aven_KO_4F_3


title: E16_MS101_121 (SingleCell389)

description: Cynomolgus Embryo at E16


title: CRAC_Skiv2l_6


title: CRAC_Xrn1_1


title: E16_MS101_120 (SingleCell388)

description: Cynomolgus Embryo at E16


title: CRAC_Skiv2l_HAR5


title: CRAC_Skiv2l_5


title: E16_MS101_117 (SingleCell386)

description: Cynomolgus Embryo at E16


title: CRAC_Skiv2l_3


title: CRAC_Skiv2l_HAR30


title: CRAC_Skiv2l_2

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


title: E16_MS101_129 (SingleCell395)

description: Cynomolgus Embryo at E16


title: E16_MS101_114 (SingleCell385)

description: Cynomolgus Embryo at E16


title: E16_MS101_119 (SingleCell387)

description: Cynomolgus Embryo at E16


title: CRAC_Skiv2l_4


title: E16_MS101_112 (SingleCell383)

description: Cynomolgus Embryo at E16


title: E16_MS101_113 (SingleCell384)

description: Cynomolgus Embryo at E16


title: CRAC_Skiv2l_1


title: E16_MS101_109 (SingleCell381)

description: Cynomolgus Embryo at E16


title: E16_MS101_111 (SingleCell382)

description: Cynomolgus Embryo at E16


title: E16_MS101_108 (SingleCell380)

description: Cynomolgus Embryo at E16


title: E16_MS101_107 (SingleCell379)

description: Cynomolgus Embryo at E16


title: CRAC_Mtr4_6


title: CRAC_Rps10_1


title: CRAC_Mtr4_3


title: E16_MS101_106 (SingleCell378)

description: Cynomolgus Embryo at E16


title: E16_MS101_105 (SingleCell377)

description: Cynomolgus Embryo at E16


title: E16_MS101_103 (SingleCell376)

description: Cynomolgus Embryo at E16


title: E16_MS101_101 (SingleCell375)

description: Cynomolgus Embryo at E16


title: CRAC_rRNA_control_2


title: CRAC_Mtr4_5


title: E16_MS101_095 (SingleCell371)

description: Cynomolgus Embryo at E16


title: E16_MS101_094 (SingleCell370)

description: Cynomolgus Embryo at E16


title: CRAC_Aven_CHX30

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


title: CRAC_Aven_1


title: E16_MS101_089 (SingleCell367)

description: Cynomolgus Embryo at E16


title: CRAC_Aven_3

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


title: E16_MS101_088 (SingleCell366)

description: Cynomolgus Embryo at E16


title: E16_MS101_090 (SingleCell368)

description: Cynomolgus Embryo at E16


title: CRAC_Skiv2l_CHX5


title: E16_MS101_098 (SingleCell373)

description: Cynomolgus Embryo at E16


title: E16_MS101_096 (SingleCell372)

description: Cynomolgus Embryo at E16


title: E16_MS101_093 (SingleCell369)

description: Cynomolgus Embryo at E16


title: CRAC_Aven_2


title: E16_MS101_086 (SingleCell364)

description: Cynomolgus Embryo at E16


title: E16_MS101_087 (SingleCell365)

description: Cynomolgus Embryo at E16


title: E16_MS101_085 (SingleCell363)

description: Cynomolgus Embryo at E16


title: CRAC_Skiv2l_CHX30


title: Auxin_M_IP

city: Boston

first: Michael

middle: Demian

last: Blower

state: MA

address: 185 Cambridge St CPZN-7250

department: Molecular Biology

country: USA

institute: Massachusetts General Hospital

library_source: genomic

data_processing:
basecalls were performed using Illumina Hi-Seq control software
reads were aligned to  hg38 using Bowtie2
reads were counted against Ensembl gene models using Cufflinks or analyzed using ngs.plot
Genome_build: hg38
Supplementary_files_format_and_content: processed data files were created by merging gene-fpkm counts for all 8 libraries.

molecule: genomic DNA

organism: Homo sapiens

source_name: HeLa S3 cells with a homozygous knock-in of the AID tag to the WAPL gene

extract_protocol:
G2 and metaphase arrested cells were collected and fixed in 1xPBS containing 1% formaldehyde for 10 minutes. Glycine was added to a final concentration of 0.125M to stop cross-linking and cells washed in cold 1xPBS. Intact nuclei from ~ 5x106 cells were extracted with 1ml of Farnham lysis buffer and finally resuspended into 300ml of RIPA buffer. Nuclei were fragmented using a Qsonica sonicator for 5 minutes (30 seconds on/off, 40%). The efficiency of fragmentation was measured as the enrichment of ~ 300 bp fragments, tested on an agarose gel, and the DNA quantified in a Biodrop. To perform immunoprecipitations Dynabeads M-280 Sheep Anti-Rabbit (Invitrogen 11203D) were coupled to 5 mg of pS2 or IgG rabbit antibodies and incubated with 25 mg of fragmented chromatin samples overnight at 4°. Beads were then washed with LiCl wash buffer and eluted in 200 ml of IP elution buffer. Cross-link was reversed by incubating beads at 65° for 1 hour, supernatant (DNA) recovered after centrifugation and incubated at 65° for 14h. In parallel, input samples (25 mg of fragmented chromatin) were incubated at 65° for 14h and then purified using QIAquick PCR Purification Kit (QIAGEN).  For ChIP-seq, DNA recovered from pS2 IP and ~ 10 ng of sonicated INPUTs were used for library preparation
NEBNext ChIP-Seq kit (NEB6240S)

treatment_protocol: synchronization: Cells were arrested in G1/S with 2mM thymidine for 16 hours, released into fresh media for 3 hours, followed by 100nM nocodazole.

value: HeLa S3

tag: genotype/variation

tag: cell cycle phase

value: M

tag: chip antibody


title: E16_MS101_084 (SingleCell362)

description: Cynomolgus Embryo at E16


title: Mock_M_IP

treatment_protocol: synchronization: Cells were arrested in G1/S with 2mM thymidine for 16 hours, released into fresh media for 3 hours, followed by 10uM RO3306.

value: homozygous knock-in of the AID tag to the WAPL gene


title: E16_MS101_083 (SingleCell361)

description: Cynomolgus Embryo at E16


title: E16_MS101_100 (SingleCell374)

description: Cynomolgus Embryo at E16


title: E16_MS101_082 (SingleCell360)

description: Cynomolgus Embryo at E16


title: E16_MS101_081 (SingleCell359)

description: Cynomolgus Embryo at E16


title: Mock_G2_IP


title: Auxin_M_input

value: none (input)


title: E16_MS101_079 (SingleCell357)

description: Cynomolgus Embryo at E16


title: Mock_M_input


title: Auxin_G2_IP


title: E16_MS101_080 (SingleCell358)

description: Cynomolgus Embryo at E16


title: Auxin_G2_input


title: Mock_G2_input


title: E16_MS101_077 (SingleCell356)

description: Cynomolgus Embryo at E16


title: E14_MS132_243 (SingleCell348)

description: Cynomolgus Embryo at E14

source_name: Cynomolgus Embryo at E14


title: Auxin_G1_1

data_processing:
basecalls were performed using Illumina Hi-Seq control software
reads were aligned to  hg38 using Tophat
reads were counted against Ensembl gene models or GeneHancer Enhancer gene models using StringTie. Aligned reads were also counted against Ensembl hg38 gene models using htseq. Read counts were compared using EdgeR.
Genome_build: hg38
Supplementary_files_format_and_content: processed data files were created by merging gene-fpkm or gene counts for all 6 libraries.

extract_protocol:
labeling: Nascent transcripts were labeled by incubation with 0.25mM EU for 2 hours following release from nocodazole.
10^6 cells were collected by certrifugation and lysed by addition of Trizol. rRNA was depleted from 5ug of total RNA using the Ribozero kit. EU-labeled RNA was biotinylated and recovered using the Click-IT EU-labeled RNA was converted to directional sequencing libraries using NEBNext Ultra Directional RNA Library Prep Kit.
NEBNext Ultra Directional RNA Library Prep Kit

treatment_protocol: synchronization: Cells were arrested in G1/S with 2mM thymidine for 16 hours, released into fresh media for 3 hours, followed by 0.1uM nocodazole for 12 hours. Released into fresh media containing EU for 2 hours.


title: E16_MS101_075 (SingleCell354)

description: Cynomolgus Embryo at E16


title: E14_MS127_155 (SingleCell345)

description: Cynomolgus Embryo at E14


title: E14_MS132_238 (SingleCell347)

description: Cynomolgus Embryo at E14


title: E16_MS101_074 (SingleCell353)

description: Cynomolgus Embryo at E16


title: Auxin_G1_2


title: Mock_G1_1


title: E14_MS127_154 (SingleCell344)

description: Cynomolgus Embryo at E14


title: E16_MS101_076 (SingleCell355)

description: Cynomolgus Embryo at E16


title: Mock_G1_3


title: E14_MS127_156 (SingleCell346)

description: Cynomolgus Embryo at E14


title: Mock_G1_2


title: Auxin_M_2

data_processing:
basecalls were performed using Illumina Hi-Seq control software
reads were aligned to  hg38 using Tophat
reads were counted against Ensembl gene models or GeneHancer Enhancer gene models using StringTie
Genes with a FPKM of 0 in any library were eliminated.
Genome_build: hg38
Supplementary_files_format_and_content: processed data files were created by merging gene-fpkm counts for all 8 libraries and removing genes with low read counts as described above.

extract_protocol:
10^6 cells were collected by certrifugation and lysed by addition of Trizol. rRNA was depleted from 5ug of total RNA using the Ribozero kit. EU-labeled RNA was biotinylated and recovered using the Click-IT EU-labeled RNA was converted to directional sequencing libraries using NEBNext Ultra Directional RNA Library Prep Kit.
NEBNext Ultra Directional RNA Library Prep Kit

tag: synchronization

value: Cells were arrested in G1/S with 2mM thymidine for 16 hours, released into fresh media for 3 hours, followed by 100nM nocodazole.

tag: labeling

value: Nascent transcripts were labeled by incubation with 0.25mM EU for 12 hours during nocodazole arrest.


title: Auxin_M_1


title: Mock_M_2


title: E14_MS127_153 (SingleCell343)

description: Cynomolgus Embryo at E14


title: E14_MS127_152 (SingleCell342)

description: Cynomolgus Embryo at E14


title: E14_MS127_151 (SingleCell341)

description: Cynomolgus Embryo at E14


title: E14_MS127_150 (SingleCell340)

description: Cynomolgus Embryo at E14


title: Auxin_G2_2

value: Cells were arrested in G1/S with 2mM thymidine for 16 hours, released into fresh media for 3 hours, followed by 10uM RO3306.

value: Nascent transcripts were labeled by incubation with 0.25mM EU for 12 hours during G2 arrest.


title: Auxin_G1_3


title: E14_MS127_149 (SingleCell339)

description: Cynomolgus Embryo at E14


title: Auxin_G2_1


title: E14_MS127_148 (SingleCell338)

description: Cynomolgus Embryo at E14


title: Mock_G2_1


title: E14_MS127_147 (SingleCell337)

description: Cynomolgus Embryo at E14


title: Mock_G2_2


title: E14_MS118_145 (SingleCell336)

description: Cynomolgus Embryo at E14


title: E14_MS118_143 (SingleCell335)

description: Cynomolgus Embryo at E14


title: E14_MS118_142 (SingleCell334)

description: Cynomolgus Embryo at E14


title: Root_plusSalt_plusDMS_rep2

city: University Park

first: Zhao

last: SU

state: PA

address: 354 N. Frear

department: Biology

institute: The Penn State University

data_processing:
Library strategy: Structure-seq2
Normalization of RT stop counts. For each transcript, the RT stop counts on each nucleotide is incremented by 1 and then taken natural log (ln), followed by normalization by the transcript’s abudance and length
Calculation of DMS reactivity. The raw DMS reactivity is calculated by subtracting the normalized RT stop counts between  (+) DMS and (-) DMS libraries
Perform normalization on the raw DMS reactivity θ(i) of all the nucleotides on all the transcripts to obtain the derived DMS reactivity of each nucleotide.
Normalization of DMS reactivities between conditions to obtain the final reactivity.
Genome_build: ensemblgenomes_Oryza_sativa_release_31
Supplementary_files_format_and_content: excel file with DMS reactivity change data

organism: Arabidopsis thaliana

source_name: Root

growth_protocol: Arabidopsis accession Col-0 was used in this study. Sterilized Arabidopsis seeds were spread on agar plates containing 1⁄2 Murashige and Skoog (MS) medium with 1% sucrose and 0.8% agar (A1296; Sigma, St. Louis, MO, USA) and stratified at 4 °C in the dark for 2 days. After stratification, the plates were transferred to the growth chamber and grown vertically for 10 d. The condition of growth chamber was set as temperature of 21 °C with an 8 h light/16 h dark photoperiod with light intensity of 150 mol m−2 s−1. Then the plants were transferred to the hydroponic setup and grown hydroponically for another 12 days. The hydroponic growth condition is 1⁄4 strength Hoagland’s solution (0.25 mM KH2PO4, 3.71 µM FeNa-EDTA, 0.5 mM MgSO4, 1.26 mM KNO3, 1.26 mM Ca(NO3)2, 11.56 µM H3BO3, 2.29 µM MnCl2, 0.20 µM ZnCl2, 0.073 µM CuCl2, 0.026 µM Na2MoO4). Solutions were changed twice per week.

extract_protocol:
Total RNA for the 24 individual biological samples was obtained in a chemical fume hood using the NucleoSpin RNA Plant kit. For each sample, 300 µg total RNA comprised the starting material for two rounds of poly(A) selection using the Poly(A)Purist MAG Kit, in order to obtain high purity mRNA for library construction.
Reverse transcription was performed using SuperScript III First-Strand Synthesis System kit using the same RT primer as previously used (1): 5'CAGACGTGTGCTCTTCCGATCNNNNNN3' which is a fusion of a random hexamer and an Illumina TruSeq Adapter. The first-strand cDNA was size-selected above 52 nt on 8M urea 10% polyacrylamide gels to remove excess RT primer and increase the ligation efficiency in the next step. After recovering cDNA using the crush-soak method, the cDNA was dissolved in 6 µL RNase-free water. Ligation was performed using T4 DNA ligase which ligated the 3'end of the cDNA to a low bias single-stranded DNA linker /5Phos/TGAAGAGCCTAGTCGCTGTTCANNNNNNCTGCCCATAGAG/3SpC3/ where the underlined sequence can forms a hairpin structure and the random hexamer can then hybridize to any cDNA fragment. Reagents were added into the cDNA solution as follows: 2 µL 10x buffer, 2 µL 5M betaine, 1 µL 100 µM linker DNA, 8 µL 50% PEG8000, 1 µL T4 DNA ligase (400U/µL). The ligation was performed at 16°C for 6 h and then 30°C for 6 h, and the enzyme was then deactivated at 65°C for 15 min. The ligation product was size selected above 90 nts on 8M urea 10% polyacrylamide gels to remove extra single-stranded linker DNA and a 67 nt ligation byproduct, consisting of one copy of the hexamer and one copy of the linker DNA. After recovery using the crush-soak method, the purified ligation product was dissolved in 10 µL RNase-free water. PCR amplification was performed using a primer specific to the single-stranded linker DNA and fused with an Illumina TruSeq Universal Adapters.

treatment_protocol: After a total of 24 days of growth, half of the plants were exposed to salt stress by addition of 100 mM NaCl (final concentration) to the Hoagland’s solution, while the rest of the plants were maintained in the original solution. The treatment lasted 48 hrs. After the salt treatment, the plants were treated with DMS as described in a subsequent section below.

value: Root

tag: ecotype

tag: age

tag: dms treatment

value: +DMS

tag: salt treatment

value: +Salt


title: E14_MS118_140 (SingleCell332)

description: Cynomolgus Embryo at E14


title: Root_plusSalt_plusDMS_rep1

tag: tissue


title: Root_plusSalt_plusDMS_rep3


title: Root_minusSalt_plusDMS_rep3

value: -Salt


title: E14_MS118_141 (SingleCell333)

description: Cynomolgus Embryo at E14

value: not applicable


title: E14_MS118_137 (SingleCell330)

description: Cynomolgus Embryo at E14

value: embryo


title: Mock_M_1


title: Root_minusSalt_plusDMS_rep1


title: Root_minusSalt_plusDMS_rep2


title: Root_plusSalt_minusDMS_rep3

value: -DMS


title: E14_MS118_134 (SingleCell328)

description: Cynomolgus Embryo at E14


title: Root_plusSalt_minusDMS_rep2


title: E14_MS118_135 (SingleCell329)

description: Cynomolgus Embryo at E14


title: Root_plusSalt_minusDMS_rep1


title: Root_minusSalt_minusDMS_rep3


title: E14_MS118_133 (SingleCell327)

description: Cynomolgus Embryo at E14


title: Root_minusSalt_minusDMS_rep1


title: Root_minusSalt_minusDMS_rep2


title: E14_MS118_132 (SingleCell326)

description: Cynomolgus Embryo at E14


title: E14_MS118_130 (SingleCell325)

description: Cynomolgus Embryo at E14


title: E14_MS118_129 (SingleCell324)

description: Cynomolgus Embryo at E14


title: E14_MS118_139 (SingleCell331)

description: Cynomolgus Embryo at E14


title: Shoot_plusSalt_plusDMS_rep2

source_name: Shoot

value: Shoot


title: E14_MS118_087 (SingleCell323)

description: Cynomolgus Embryo at E14


title: Shoot_plusSalt_plusDMS_rep1


title: Shoot_minusSalt_plusDMS_rep3


title: E14_MS118_081 (SingleCell321)

description: Cynomolgus Embryo at E14


title: Shoot_minusSalt_plusDMS_rep2


title: E14_MS118_083 (SingleCell322)

description: Cynomolgus Embryo at E14


title: Shoot_plusSalt_minusDMS_rep2


title: E14_MS118_075 (SingleCell319)

description: Cynomolgus Embryo at E14


title: Shoot_minusSalt_minusDMS_rep3


title: Shoot_plusSalt_minusDMS_rep3


title: E14_MS118_071 (SingleCell315)

description: Cynomolgus Embryo at E14


title: E14_MS118_073 (SingleCell317)

description: Cynomolgus Embryo at E14


title: E14_MS118_074 (SingleCell318)

description: Cynomolgus Embryo at E14


title: E14_MS118_072 (SingleCell316)

description: Cynomolgus Embryo at E14


title: E14_MS118_070 (SingleCell314)

description: Cynomolgus Embryo at E14


title: Shoot_minusSalt_plusDMS_rep1


title: Shoot_minusSalt_minusDMS_rep1


title: E14_MS118_067 (SingleCell313)

description: Cynomolgus Embryo at E14


title: Shoot_minusSalt_minusDMS_rep2


title: DN3_DKO_rep3 [DT3]

city: Chicago

first: Barbara

middle: L

last: Kee

state: Illinois

address: 924 E57th st

department: Pathology

institute: The University of Chicago

description:
DT3_S3
processed data file: DN3.edgeR.txt

data_processing:
Raw sequence reads were trimmed using Trimmomatic v 0.33 (TRAILING:30 MINLEN:20)
Trimmed reads were aligned to mouse genome assembly mm10 with Tophat v 2.1.0.
Reads were assigned to genes using the htseq-count tool from HTSeq v 0.6.1 and gene annotations from Ensembl release 78.
The R package EdgeR was used to normalize the gene counts and to calculate differential expression statistics for each gene for each pairwise comparison of sample groups. Genes were considered differentially expressed if the |fold change| >= 2 and FDR < 0.05.
Genome_build: mm10
Supplementary_files_format_and_content: normalized abundance measurements provided as edgeR output files

extract_protocol:
Total RNA was isolated using RNAeasy MicroKit (Qiagen) according to manufacturer’s recommendations.
Libraries were constructed using Nugen’s Ovation Ultralow Library systems and were subsequently subjected to 76 cycles of NextSeq500 sequencing.

treatment_protocol: pro-B (CD19+B220+CD43+), DN3 (Lin-CD25+CD117-) were sorted from Cdkn2a-/- Ezh2fl/fl Il7racre/+ mice and Cdkn2a-/- Il7racre/+ controls into RLT buffer.  Three independent samples per genotype were assessed.

tag: strain background

value: Cdkn2a-/- Ezh2-fl/fl Il7ra-cre/+

value: Thymus


title: Shoot_plusSalt_minusDMS_rep1


title: DN3_DKO_rep1 [DT1]

description:
DT1_S5
processed data file: DN3.edgeR.txt


title: Shoot_plusSalt_plusDMS_rep3


title: DN3_DKO_rep2 [DT2]

description:
DT2_S1
processed data file: DN3.edgeR.txt


title: E14_MS118_066 (SingleCell312)

description: Cynomolgus Embryo at E14


title: E14_MS117_109 (SingleCell310)

description: Cynomolgus Embryo at E14


title: DN3_Cntrl_rep3 [CT3]

description:
CT3_S2
processed data file: DN3.edgeR.txt


title: E13_MS134_059 (SingleCell309)

description: Cynomolgus Embryo at E13

source_name: Cynomolgus Embryo at E13


title: E14_MS118_076 (SingleCell320)

description: Cynomolgus Embryo at E14


title: DN3_Cntrl_rep2 [CT2]

description:
CT2_S6
processed data file: DN3.edgeR.txt


title: E13_MS134_058 (SingleCell308)

description: Cynomolgus Embryo at E13


title: DN3_Cntrl_rep1 [CT1]

description:
CT1_S4
processed data file: DN3.edgeR.txt


title: E13_MS134_057 (SingleCell307)

description: Cynomolgus Embryo at E13


title: E13_MS133_056 (SingleCell306)

description: Cynomolgus Embryo at E13


title: proB_DKO_rep2 [DB2]

description:
DB2_S3
processed data file: proB.edgeR.txt

value: Bone Marrow


title: proB_DKO_rep3 [DB3]

description:
DB3_S1
processed data file: proB.edgeR.txt


title: E13_MS133_055 (SingleCell305)

description: Cynomolgus Embryo at E13


title: proB_DKO_rep1 [DB1]

description:
DB1_S5
processed data file: proB.edgeR.txt


title: E13_MS133_054 (SingleCell304)

description: Cynomolgus Embryo at E13


title: proB_Cntrl_rep3 [CB3]

description:
CB3_S2
processed data file: proB.edgeR.txt


title: E14_MS118_001 (SingleCell311)

description: Cynomolgus Embryo at E14


title: E13_MS132_251 (SingleCell302)

description: Cynomolgus Embryo at E13


title: proB_Cntrl_rep2 [CB2]

description:
CB2_S4
processed data file: proB.edgeR.txt


title: E13_MS133_053 (SingleCell303)

description: Cynomolgus Embryo at E13


title: proB_Cntrl_rep1 [CB1]

description:
CB1_S6
processed data file: proB.edgeR.txt


title: E13_MS132_248 (SingleCell300)

description: Cynomolgus Embryo at E13


title: E13_MS132_234 (SingleCell299)

description: Cynomolgus Embryo at E13


title: E13_MS132_214 (SingleCell296)

description: Cynomolgus Embryo at E13


title: Cyba-KO 2

city: Salamanca

first: Ángel

last: Hernández-Hernández

state: Salamanca

address: Plaza Doctores de la Reina s/n, Edificio Departamental, Lab 122

department: Biochemistry and Molecular Biology

country: Spain

institute: University of Salamanca (USAL)

data_processing:
Illumina NextSeq 500 software was used for basecalling
Samples were aligned with Salmon algorithm (Patro et al. 2017) setting following parameters: single-end, default parameters.
Differential Expression analysis was performed with DESeq2 (Love, Anders, and Huber 2014). It was executed with following parameters: test: Wald; fitType: parametric. It found 53 significant genes for a cutoff of 0.05.
Protocol executed by RaNA-seq (Bioinformatic tool from de University of Salamanca, http://ranaseq.usal.es).
Genome_build: GRCm38
Supplementary_files_format_and_content: excel files including total counts and TPM values for each sample

source_name: Bone marrow lineage negative cells

extract_protocol:
Bone marrow cells were extracted by flushing femurs, tibias and hip bones from mice, and extracts were filtered and washed with phosphate-buffered saline (PBS). Red blood cells were lysated with ammonium chloride buffer at 4ºC and samples were washed in PBS again. Lineage negative cells were purified with the Lineage Cell Depletion Kit for mice (Miltenyi Biotec), according to the manufacturer´s instructions. Total RNA was harvested from Lineage negative cells using Illustra RNAspin Mini Isolation Kit (GE Healthcare).
Illumina TruSeq RNA Library Preparation Kit v2 was used with 2 ug of total RNA for the construction of sequencing libraries.

tag: strain

tag: genotype

value: Cyba -/-


title: E13_MS132_229 (SingleCell298)

description: Cynomolgus Embryo at E13

tag: cell line

tag: cell type


title: E13_MS132_250 (SingleCell301)

description: Cynomolgus Embryo at E13

tag: developmental stage


title: Cyba-KO 1


title: WT 1

value: Wild type


title: E13_MS132_225 (SingleCell297)

description: Cynomolgus Embryo at E13


title: E13_MS132_205 (SingleCell294)

description: Cynomolgus Embryo at E13


title: E13_MS132_204 (SingleCell293)

description: Cynomolgus Embryo at E13


title: E13_MS132_032 (SingleCell291)

description: Cynomolgus Embryo at E13


title: E13_MS132_202 (SingleCell292)

description: Cynomolgus Embryo at E13


title: E13_MS132_031 (SingleCell290)

description: Cynomolgus Embryo at E13


title: E13_MS132_030 (SingleCell289)

description: Cynomolgus Embryo at E13


title: E13_MS132_029 (SingleCell288)

description: Cynomolgus Embryo at E13


title: E13_MS132_028 (SingleCell287)

description: Cynomolgus Embryo at E13


title: E13_MS132_027 (SingleCell286)

description: Cynomolgus Embryo at E13


title: E13_MS132_026 (SingleCell285)

description: Cynomolgus Embryo at E13


title: WT 2


title: E13_MS132_025 (SingleCell284)

description: Cynomolgus Embryo at E13


title: E13_MS132_021 (SingleCell280)

description: Cynomolgus Embryo at E13


title: E13_MS132_206 (SingleCell295)

description: Cynomolgus Embryo at E13


title: E13_MS132_020 (SingleCell279)

description: Cynomolgus Embryo at E13


title: E20_MS008_086 (SingleCell576)

description: Cynomolgus Embryo at E20

source_name: Cynomolgus Embryo at E20


title: E13_MS130_065 (SingleCell273)

description: Cynomolgus Embryo at E13


title: E13_MS130_071 (SingleCell274)

description: Cynomolgus Embryo at E13


title: E13_MS132_013 (SingleCell275)

description: Cynomolgus Embryo at E13


title: E13_MS130_064 (SingleCell272)

description: Cynomolgus Embryo at E13


title: E13_MS132_017 (SingleCell276)

description: Cynomolgus Embryo at E13


title: E16_MS083_007 (SingleCell351)

description: Cynomolgus Embryo at E16


title: E13_MS132_018 (SingleCell277)

description: Cynomolgus Embryo at E13


title: E13_MS130_063 (SingleCell271)

description: Cynomolgus Embryo at E13


title: E16_MS009_038 (SingleCell350)

description: Cynomolgus Embryo at E16


title: E13_MS130_050 (SingleCell268)

description: Cynomolgus Embryo at E13


title: E13_MS130_057 (SingleCell269)

description: Cynomolgus Embryo at E13


title: E13_MS130_048 (SingleCell267)

description: Cynomolgus Embryo at E13


title: E13_MS083_050 (SingleCell199)

description: Cynomolgus Embryo at E13


title: E13_MS083_028 (SingleCell198)

description: Cynomolgus Embryo at E13


title: E13_MS132_024 (SingleCell283)

description: Cynomolgus Embryo at E13


title: E13_MS132_023 (SingleCell282)

description: Cynomolgus Embryo at E13


title: E13_MS130_047 (SingleCell266)

description: Cynomolgus Embryo at E13


title: E13_MS010_053 (SingleCell197)

description: Cynomolgus Embryo at E13


title: E13_MS132_022 (SingleCell281)

description: Cynomolgus Embryo at E13


title: E13_MS130_046 (SingleCell265)

description: Cynomolgus Embryo at E13


title: E09_MS068_087 (SingleCell182)

description: Cynomolgus Embryo at E09

source_name: Cynomolgus Embryo at E09


title: E13_MS130_045 (SingleCell264)

description: Cynomolgus Embryo at E13


title: E16_MS083_012 (SingleCell352)

description: Cynomolgus Embryo at E16


title: E13_MS132_019 (SingleCell278)

description: Cynomolgus Embryo at E13


title: E13_MS130_044 (SingleCell263)

description: Cynomolgus Embryo at E13


title: E13_MS130_059 (SingleCell270)

description: Cynomolgus Embryo at E13


title: E09_MS003_096 (SingleCell181)

description: Cynomolgus Embryo at E09


title: E07_MS068_033 (SingleCell073)

description: Cynomolgus Embryo at E07

source_name: Cynomolgus Embryo at E07


title: E16_MS008_070 (SingleCell349)

description: Cynomolgus Embryo at E16


title: E07_MS008_047 (SingleCell072)

description: Cynomolgus Embryo at E07


title: E07_MS003_073 (SingleCell070)

description: Cynomolgus Embryo at E07


title: E13_MS130_025 (SingleCell261)

description: Cynomolgus Embryo at E13


title: E13_MS130_023 (SingleCell259)

description: Cynomolgus Embryo at E13


title: E07_MS003_066 (SingleCell068)

description: Cynomolgus Embryo at E07


title: E07_MS003_074 (SingleCell071)

description: Cynomolgus Embryo at E07


title: E13_MS130_026 (SingleCell262)

description: Cynomolgus Embryo at E13


title: E13_MS130_020 (SingleCell257)

description: Cynomolgus Embryo at E13


title: E13_MS130_022 (SingleCell258)

description: Cynomolgus Embryo at E13


title: E07_MS003_069 (SingleCell069)

description: Cynomolgus Embryo at E07


title: E13_MS130_019 (SingleCell256)

description: Cynomolgus Embryo at E13


title: E07_MS003_051 (SingleCell067)

description: Cynomolgus Embryo at E07


title: E07_MS003_039 (SingleCell065)

description: Cynomolgus Embryo at E07


title: E13_MS130_017 (SingleCell254)

description: Cynomolgus Embryo at E13


title: E06_MS003_037 (SingleCell046)

description: Cynomolgus Embryo at E06

source_name: Cynomolgus Embryo at E06


title: E06_MS003_036 (SingleCell045)

description: Cynomolgus Embryo at E06


title: E13_MS127_191 (SingleCell253)

description: Cynomolgus Embryo at E13


title: E13_MS130_018 (SingleCell255)

description: Cynomolgus Embryo at E13


title: E13_MS127_190 (SingleCell252)

description: Cynomolgus Embryo at E13


title: E13_MS130_024 (SingleCell260)

description: Cynomolgus Embryo at E13


title: E06_MS003_035 (SingleCell044)

description: Cynomolgus Embryo at E06


title: E06_MS003_031 (SingleCell042)

description: Cynomolgus Embryo at E06


title: E13_MS127_188 (SingleCell251)

description: Cynomolgus Embryo at E13


title: E06_MS003_032 (SingleCell043)

description: Cynomolgus Embryo at E06


title: E13_MS127_171 (SingleCell249)

description: Cynomolgus Embryo at E13


title: E13_MS127_069 (SingleCell245)

description: Cynomolgus Embryo at E13


title: E13_MS127_062 (SingleCell244)

description: Cynomolgus Embryo at E13


title: E13_MS127_187 (SingleCell250)

description: Cynomolgus Embryo at E13


title: E13_MS127_167 (SingleCell248)

description: Cynomolgus Embryo at E13


title: E13_MS118_128 (SingleCell239)

description: Cynomolgus Embryo at E13


title: E13_MS127_166 (SingleCell247)

description: Cynomolgus Embryo at E13


title: E13_MS118_125 (SingleCell237)

description: Cynomolgus Embryo at E13


title: E13_MS118_147 (SingleCell241)

description: Cynomolgus Embryo at E13


title: E13_MS127_144 (SingleCell246)

description: Cynomolgus Embryo at E13


title: E13_MS118_121 (SingleCell236)

description: Cynomolgus Embryo at E13


title: E13_MS127_049 (SingleCell242)

description: Cynomolgus Embryo at E13