title: SmallSeqBatch - MNs - H9 WT - rep2

city: Cleveland

first: Ashleigh

last: Schaffer

state: OH

address: 2109 Adelbert Road

department: Department of Genetics and Genome Sciences and Center for RNA Science and Therapeutics

institute: Case Western Reserve University

description: Mature d28 motor neurons differentiated from H9 ESCs, higher MN purity differentiation, lower cell seeding density, low untemplated adenosine library. Used only for alternative polyadenylation (APA) analysis.

data_processing:
Library strategy: PAC-Seq
For mRNA 3’ end sequencing analysis, two batches of PAC-seq were run. One batch was a collection of RNA from 16 human stem cell-derived motor neuron cultures from the PCH10 index patient family and H9 genetic backgrounds ("large batch"). A smaller batch containing H9 and H9 CLP1 KO iPSC-derived motor neuron RNA samples, with two replicates each, was also run ("small batch"). The larger PAC-seq batch showed ample polyadenosine homopolymeric stretches within the 3’-end of its reads, whereas the smaller batch did not. As such, only samples from the larger batch were used to construct in-house annotation databases of human and mouse motor neuron poly(A) sites.
For both batches of PAC-seq, each sample’s raw reads underwent pre-alignment trimming which ensured polyadenosine stretches at the 3’-end of reads remained intact. Reads had any 2-color sequencing chemistry artifacts trimmed with Cutadapt (Martin, 2011) using settings -a G{150} -e 0.4. Any reads smaller than 40 bp after trimming were filtered out.
In the larger batch, 12 nucleotide unique molecular identifiers (UMIs) added in library preparation were removed from the 5’ end of each read and appended to the read header using umi_tools (Smith et al., 2017).
Processed reads from both sequencing batches were aligned to the hg38 genome build with STAR (Dobin et al., 2013) using an alignment strategy which only performs softclipping on the 3’-end of reads (--alignEndsType Extend5pOfRead1).  Because untemplated poly(A) tails could cover large portions of input reads, STAR was also set to be less strict with the minimum proportion of the read aligned (--outFilterMatchNminOverLread 0.3 and --outFilterScoreMinOverLread 0.3). This strategy optimized for alignments which ended directly at the poly(A) site but still contained some poly(A) tail sequence within the softclipped data.
A database of poly(A) sites used by human motor neurons was constructed only from the larger batch of PAC-seq samples' alignments. These alignments were filtered with an in-house script to remove any entries with no softclipping before deduplication using umi_tools. A final filtering step performed with in-house scripts inspected the actual sequence data that was softclipped and removed any read alignments which did not show enough untemplated adenosines to meet the standards followed by Gencode for their poly(A) site annotations (Derti et al., 2012). Specifically, each read’s softclipped sequence needed to contain 1) greater than 66% adenosine content and 2) at least 3 untemplated adenosines. This filtering step was made more strict for sites where sequence just upstream of the softclip indicated that the read came from an internal priming hotspot, in which case the sum of terminal adenosines must also exceed the length of the oligo d(T) primer.
Using these 16 high-confidence alignments, each sample had its unique poly(A) sites defined by taking slices of read coverage greater than a minimum read threshold, keeping the most 3’-end nucleotide of each slice region, then reducing the read threshold repeatedly, keeping the higher coverage slice region when there were overlaps. This coverage slicing process was repeated when merging each sample’s unique poly(A) sites to output a final set of sites that showed at least 3 reads in at least 2 samples, and were at least 100 bp apart.
Providing each poly(A) site with an assigned gene and intragenic feature followed logic that replicates how one annotates poly(A) sites by hand. Each poly(A) site was checked for overlap or proximity to specific intragenic features in order of likelihood, and removed from the pool for annotation when a probable match was found. These features in order were transcript ends, 3’ UTRs, terminal exons, regions up to 10 kb downstream of transcript ends, introns, CDS, 5’ UTRs, and exons, respectively. Any site which had more than one potential match in a step would be assigned to the longest transcript and its corresponding gene. Determining which intragenic feature of an assigned gene that a poly(A) site was part of followed the rules applied to PolyA_DB (Wang et al., 2018) with the change that we used Gencode gene and transcript annotations v37 for human (Frankish A, et al., 2018).
Poly(A) site counts were acquired using countOverlaps in the GenomicRanges R package (Lawrence et al., PLoS Computational Biology, 2013), with inputs of either fully-filtered per-sample alignments as described above, or in the case of samples in the smaller sequencing batch, unfiltered alignments were used as count inputs and counts were mathematically adjusted before use in DEXSeq to reflect the site-specific proportion of reads that passed filters in the 16 other samples. Any genes with only one annotated poly(A) site were excluded from analysis.
Genome_build: hg38
Supplementary_files_format_and_content: bedGraph files were generated by taking the pileup of the 3'-most 20bp of either filtered high confidence aligned reads ("large batch") or all aligned reads ("small batch"). These are representative of read pileup and ranges used as input for feature counting against the motor neuron-specific poly(A) site annotations generated in this study. Two bedgraphs exist for each sample: one for each strand of the genome.
Supplementary_files_format_and_content: Feature count files are tab-delimited counts of overlaps with the 3'-most 20bp of either filtered high confidence aligned reads ("large batch") or all aligned reads ("small batch") and the motor neuron-specific poly(A) site annotations generated in this study. These files were only generated for samples which 1) were from differentiations with high motor neuron purity, 2) were genotypically pure, and 3) had a clonal or technical replicate in the same sequencing batch. Feature count files from the "small batch" have been mathematically adjusted to account for the empirically determined internal priming rate at each poly(A) site by multiplying raw counts with the inverse of the site-specific internal priming rate before rounding.
Supplementary_files_format_and_content: Excel spreadsheet file is the human motor neuron-specific poly(A) site database generated in this study. Poly(A) site identifiers used for alternative polyadenylation analysis in DEXSeq employ a naming convention of Gencode v37 gene identifier, a colon, and the relative ordered position of sites within their gene from 5' to 3' relative to the + strand. Any sites with a match to an annotated poly(A) site in PolyA_DB v3.2 (proximity of 100bp or less) have their polyA_DB ID and genomic coordinates (lifted over to hg38) listed.

source_name: ESC-derived motor neurons

growth_protocol: Primary fibroblast cell lines were established from skin biopsies of affected children and an unaffected parent according to standard methods. Reprogramming of patient fibroblasts into induced pluripotent stem cells (iPSCs) was performed using the Epi5 Episomal iPSC Reprogramming Kit (ThermoFisher) with the following modifications. Electroporated fibroblasts were plated on Matrigel membrane matrix (Fisher). Reprogramming vectors were prepared in house and the medium was changed to StemFlex (ThermoFisher) at day 15. iPSC clones were selected for expansion and plated on vitronectin-coated plates. Motor neurons were differentiated from patient-derived iPSCs or H9 ESCs and their derivatives using a previously published protocol (Markmiller et a., Cell, 2018)

extract_protocol:
TRIzol
Two batches of poly(A) click-seq (PAC-Seq) libraries were constructed as described in Routh et al., Nucleic Acids Research, 2017.

value: CLP1 WT

tag: genetic background

value: H9 ESC

tag: sample usage in study

value: APA analysis only

tag: pac-seq batch

value: Small


title: SmallSeqBatch - MNs - H9 WT - rep1

status: Public on Nov 01 2021

type: SRA

country: USA

description: Mature d28 motor neurons differentiated from H9 ESCs, high MN purity differentiation, higher cell seeding density, low untemplated adenosine library. Used only for alternative polyadenylation (APA) analysis.

library_source: transcriptomic

molecule: polyA RNA

organism: Homo sapiens

tag: genotype


title: SmallSeqBatch - MNs - H9 CLP1 KO - rep1

description: Mature d28 motor neurons differentiated from H9 ESCs with complete CRISPR knockout of CLP1, higher MN purity differentiation, low untemplated adenosine library. Used only for alternative polyadenylation (APA) analysis.

value: CLP1 KO


title: SmallSeqBatch - MNs - H9 CLP1 KO - rep2


title: FaDu-Mock#2-2

type: RNA

city: Heidelberg

first: Chunxuan

last: Shao

address: Im Neuenheimer Feld 280

country: Germany

institute: German Cancer Research Center

hyb_protocol: Hybridization was performed at 58°C, in GEX-HCB buffer (Illumina Inc.) at a concentration of 150 ng cRNA/µl, unsealed in a wet chamber for 20h. Spike-in controls for low, medium and highly abundant RNAs were added, as well as mismatch control and biotinylation control oligonucleotides. Microarrays were washed once in High Temp Wash buffer (Illumina Inc.) at 55°C and then twice in E1BC buffer (Illumina Inc.) at room temperature for 5 minutes (in between washed with ethanol at room temperature). After blocking for 5 min in 4 ml of 1% (wt/vol) Blocker Casein in phosphate buffered saline Hammarsten grade (Pierce Biotechnology, Inc., Rockford, IL), array signals are developed by a 10-min incubation in 2 ml of 1 µg/ml Cy3-streptavidin (Amersham Biosciences, Buckinghamshire, UK) solution and 1% blocking solution. After a final wash in E1BC, the arrays are dried and scanned.

scan_protocol: Microarray scanning was done using an iScan array scanner (Illumina Inc.). Data extraction was done for all beads individually, and the outliers are removed when the absolute difference to the median was greater than 2.5 times MAD(2.5 Hampelís method).

data_processing:
Raw data was read and normalized via lumi (R packages), processed was then analyzed by limma incorporating the technical replicate information.   The analysis is done with R on the bead-level data which is  found in the raw data produced by the scanner (e.g. in  5282035051.7z the file 5282035051_A.txt, which holds bead  level data for all probeIDs of sample A).    All analyses are done using the following mechanisms:  1) outlier removal (see below).  2) average values over beads are means (not median).  3) statistical tests are t-tests over all beads (e.g for one      probeID over all beads of all samples of a group) in original     scale (not log2 scale).     Example:      group x is sample A,B,C      control y is sample D,E,F      to test for significance of difference between group x       and control y, we use      t.test(all_beads_of_A_B_C, all_beads_D_E_F,         alternative = two.sided,        mu = 0 )       Example:      to check for significance against background (one specific       probeID):        t.test(all_beads_of_A, all_negative_of_A, alternative=greater)       Benjamini-Hochberg correction is applied over all p-values of     the differential expression analysis.         Calculation of GeneView data:  The calculations are also done on the set of beads which belong to  a given Gene Symbol (annotation column Symbol).         Outlier removal on bead level:    Outliers are removed by taking only beads with an expression value > 20 before  using the 2.5 MADs rule. The ProbeIDs expression value is than the mean of the   remaining beads.    The remaining bead level lists of different length for each sample are randomly  filled with NAs (NA=not available) and build the bead level matrix on which quantile  normalization is performed.    MAD rule:  mad = median absolute deviation  (<http://en.wikipedia.org/wiki/Median_absolute_deviation>)  x = one bead level expression value of some specific probeID  Than:  outliers are those x where abs((x-median)/mad) > 2.5
All remaining bead level data points are than quantile   normalized. As test for significance the studentís t-test is   used on the bead expression values of the two groups of interest.   In the case of significance of expression against background we   tested for greater than all negative beads for this sample and   in the case of comparing separate groups we tested for inequality   of the means of the groups. In both cases Benjamini-Hochberg   correction was applied to the complete set of p-values of   all ProbeIDs on the chip. The average expression value is   calculated as mean of the measured expressions of beads   together with the standard deviation of the beads.

label: biotin

molecule: total RNA

source_name: cell line FaDu-Mock

label_protocol: Biotin-labeled cRNA samples for hybridization on Illumina Human Sentrix-12 BeadChip arrays (Illumina Inc.) were prepared according to manufacturer’s recommended sample labeling procedure based on the modified Eberwine protocol (Eberwine et al., Methods Enzymol, 216 (1992) 80-100). In brief, 300 ng total RNA was used for complementary DNA (cDNA) synthesis, followed by an amplification/labeling step (in vitro transcription) to synthesize biotin-labeled cRNA according to the Illumina® Total Prep™ RNA Amplification Kit (Life Technologies, Germany).  Biotin-16-UTP was purchased from Roche Applied Science, Penzberg, Germany. The cRNA was column purified according to TotalPrep RNA Amplification Kit, and eluted in 60 µl of water. Quality and quantity of cRNA was confirmed  using the RNA Nano Chip Assay on an Agilent 2100 Bioanalyzer (Agilent Technologies, Germany).

growth_protocol: FaDu cells were purchased from ATCC (HTB-43) and authentication was confirmed by the Multiplex Human Cell Line Authentication Test (Multiplexion, Germany). Generation of FaDu-mock and FaDu-shKLK6 clones was described elsewhere (Schrader et al., Mol Cancer. 2015 May 20;14:107). Stable clones were maintained in Dulbecco’ s Modified Eagle’ s Medium (Sigma, Germany) supplemented with 10 % fetal bovine serum (Sigma, Germany), 2 mM L-Glutamine (Sigma, Germany) and 50 μg/ml Penicillin-Streptomycin (Sigma, Germany) in a humidified atmosphere of 6-8 % CO2  at 37 °C.

extract_protocol: Total RNA was isolated with the RNeasy Mini Kit (Qiagen, Germany) following the manufacturer’s instruction. DNA digestion was performed with RNase-free DNAse Set (Qiagen, Germany). Quantity and quality of isolated RNA was determined with the help of the Nanodrop Spectrophotometer ND-1000 (Peqlab, Germany).

treatment_protocol: As described in Schrader et al. (Mol Cancer. 2015 May 20;14:107) FaDu cells were transfected with either a control pRS vector encoding a non-effective Hush 29-mer scrambled shRNA cassette (TR30012, OriGene Technologies, USA) or a pRS-shKLK6 plasmid (TR316673, OriGene Technologies, USA) using FuGene HD Transfection Reagent (Promega, Germany) according to the manufacturer’ s instruction. Following selection with 1.5 μg/ml Puromycin (Gibco life technologies, Germany) for one week, stable clones were isolated and KLK6 silencing was confirmed on RNA level by RQ-PCR.

tag: cell type

value: HNSCC derived cell line

tag: cell line

value: FaDu-Mock

tag: technical replicates group


title: FaDu-shKLK6#1-2

source_name: cell line FaDu-shKLK6


title: LargeSeqBatch - MNs - Unaffected parent clone 2 - JL diff

description: Mature d28 motor neurons differentiated from Unaffected parent, iPSC clone 2, lower MN purity differentiation, high untemplated adenosine library. Used only for creation of an MN-specific poly(A) site database.

source_name: iPSC-derived motor neurons

value: PCH10 index family

value: Creation of MN poly(A) site database only

value: Large


title: LargeSeqBatch - MNs - H9 Mixed CLP1 KO - JL diff - rep2

description: Mature d28 motor neurons differentiated from H9 ESCs with CRISPR knockout of CLP1, which was determined to be a mixed colony with low detectable expression of CLP1 protein, lower MN purity differentiation, high untemplated adenosine library. Used only for creation of an MN-specific poly(A) site database.

value: Mixed CLP1 KO and CLP1 WT


title: LargeSeqBatch - MNs - H9 Mixed CLP1 KO - JL diff - rep1


title: LargeSeqBatch - MNs - Unaffected parent clone 3 - AES diff

description: Mature d28 motor neurons differentiated from Unaffected parent, iPSC clone 3, higher MN purity differentiation, high untemplated adenosine library. Used for both creation of an MN-specific poly(A) site database and alternative polyadenylation (APA) analysis.

value: Creation of MN poly(A) site database, APA analysis


title: LargeSeqBatch - MNs - Unaffected parent clone 3 - JL diff

description: Mature d28 motor neurons differentiated from Unaffected parent, iPSC clone 3, lower MN purity differentiation, high untemplated adenosine library. Used only for creation of an MN-specific poly(A) site database.


title: LargeSeqBatch - MNs - H9 Mixed CLP1 KO - AES diff

description: Mature d28 motor neurons differentiated from H9 ESCs with CRISPR knockout of CLP1, which was determined to be a mixed colony with low detectable expression of CLP1 protein, higher purity MN differentiation, high untemplated adenosine library. Used only for creation of an MN-specific poly(A) site database.


title: LargeSeqBatch - MNs - H9 WT - JL diff - rep1

description: Mature d28 motor neurons differentiated from H9 ESCs, lower MN purity differentiation, high untemplated adenosine library. Used only for creation of an MN-specific poly(A) site database.


title: LargeSeqBatch - MNs - Affected child clone 2 - Mixed CRISPR correction subclone 25 - rep2

description: Mature d28 motor neurons differentiated from Affected patient, iPSC clone 2, CRISPR correction subclone 25, which was determined to be a mixed colony with homozygous and heterozygous CLP1 R140H mutation, high untemplated adenosine library. Used only for creation of an MN-specific poly(A) site database.

value: Mixed CLP1 R140H/R140H and CLP1 R140H/+


title: LargeSeqBatch - MNs - Unaffected parent clone 2 - AES diff

description: Mature d28 motor neurons differentiated from Unaffected parent, iPSC clone 2, higher MN purity differentiation, high untemplated adenosine library. Used for both creation of an MN-specific poly(A) site database and alternative polyadenylation (APA) analysis.


title: LargeSeqBatch - MNs - H9 WT - AES diff

description: Mature d28 motor neurons differentiated from H9 ESCs, higher MN purity differentiation, high untemplated adenosine library. Used only for creation of an MN-specific poly(A) site database.


title: LargeSeqBatch - MNs - Affected child clone 2 - Mixed CRISPR correction subclone 25 - rep1


title: FaDu-shKLK6#3-1


title: LargeSeqBatch - MNs - H9 WT - JL diff - rep2


title: LargeSeqBatch - MNs - Affected child clone 2 - JL diff

description: Mature d28 motor neurons differentiated from Affected patient, iPSC clone 2, lower MN purity differentiation, high untemplated adenosine library. Used only for  creation of an MN-specific poly(A) site database.


title: CD11C- CD8T 2

city: Jinan

first: An-Liang

last: Guo

address: No.44 West Wenhua Road

department: Department of Immunology

country: China

institute: School of Basic Medical Sciences, Cheeloo College of Medicine, Shandong University

data_processing:
Whole-transcriptome amplification and tagmentation-based library preparation were performed using SMART-seq2, followed by sequencing on a NextSeq 500 system (Illumina, San Diego, CA, USA).
Transcript abundance was quantified using RSEM software (v1.2.22) supported by STAR aligner software (STAR 2.5.1b) and aligned to the GRCh38 human genome.
The transcripts per million values were normalized among all samples using the upper-quantile normalization method.
Genome_build: Homo_sapiens.GRCh38.94.chr
Supplementary_files_format_and_content: Processed data files include  Sample Matrix table of FPKM and gene counts  for every gene and every sample

source_name: PBMC

extract_protocol:
Total RNA from the sorted CD11c+ and CD11c- CD8+ T cells was extracted using the PicoPure RNA Isolation Kit (Applied Biosystems, Foster City, CA, USA).
Whole transcriptome amplification and tag-based library preparation were conducted using SMART-seq2

treatment_protocol: PBMCs from 3 healthy donors were isolated using Ficoll-Paque PLUS density gradient centrifugation, CD8+ T cells were purified from PBMCs using magnetic-activated cell sorting beads(130–093-227; Miltenyi Biotec, Bergisch-Gladbach, Germany). After labeling CD8+ T cells with anti-CD3-APC/Fire750, anti-CD8-BV510, and anti-CD11c-FITC antibodies, CD11c+ and CD11c- CD8+ T cells were sorted using a MA900 Cell Sorter (Sony Biotechnology, Tokyo, Japan).

value: CD11C- CD8 T cells

tag: individual

value: healthy donor no.2


title: CD11C- CD8T 1

value: healthy donor no.1


title: CD11C- CD8T 3

value: healthy donor no.3


title: CD11C+ CD8T 1

value: CD11C+ CD8 T cells


title: LargeSeqBatch - MNs - Affected child clone 2 - AES diff

description: Mature d28 motor neurons differentiated from Affected patient, iPSC clone 2, higher MN purity differentiation, high untemplated adenosine library. Used for both creation of an MN-specific poly(A) site database and alternative polyadenylation (APA) analysis.


title: LargeSeqBatch - MNs - Affected child clone 1 - AES diff

description: Mature d28 motor neurons differentiated from Affected patient, iPSC clone 1, higher MN purity differentiation, high untemplated adenosine library. Used for both creation of an MN-specific poly(A) site database and alternative polyadenylation (APA) analysis.


title: LargeSeqBatch - MNs - Affected child clone 1 - JL diff

description: Mature d28 motor neurons differentiated from Affected patient, iPSC clone 1, lower MN purity differentiation, high untemplated adenosine library. Used only for  creation of an MN-specific poly(A) site database.


title: BBB model replicate 2

city: Indiana

first: Lane

middle: A

last: Baker

state: IN

address: Indiana University 800 E Kirkwood Ave

department: Chemistry

institute: Indiana University

data_processing:
The raw RNA-seq reads were cleaned using Trimmomatic version 0.36 (Bolger et al. 2014) setting the cutoff threshold for average base quality score at 20 over a window of 3 bases. Reads shorter than 20 bases post-trimming were excluded. (parameters: LEADING:20 TRAILING:20 SLIDINGWINDOW:3:20 MINLEN:20)
The cleaned reads were aligned to the human genome reference, GRCh38.p12 using STAR (version STAR_2.5.2b) (Dobin et al. 2013).
Individual gene expression was quantified by counting the number reads mapping to the exon regions of each of the annotated genes from gencode v.28 using featureCounts tool of subread package (version 2.0.0) (Liao et al. 2014). Those reads that mapped to antisense strand, or mapped to multiple regions on the genome or those reads that overlapped with multiple genes were excluded. (parameters: -s 2 -p -B -C)
Genome_build: GRCh38.p12
Supplementary_files_format_and_content: Tab deliminted text files containing counts of reads overlapping with exon regions of the genes.

source_name: brain endothelial cells + brain astrocytes

growth_protocol: Endothelial cells and astrocytes were maintained in endothelial medium and astrocyte medium, respectively at 37 degree Celsius.

extract_protocol:
Cells were lysed by Trizol reagent followed by addition of chloroform. After centrifuge, the aqueous phase was obtained and ispropyl alcohol was added to the aqueous phase to precipitate the RNA. The RNA pellet was washed by 75% ethanol and the dissolved in RNase-free water.
RNA libraries were prepared for sequencing using standard Illumina protocols

treatment_protocol: Cells were washed with PBS prior to extraction.

value: Induced pluripotent stem cell-derived brain endothelial cells and astrocytes


title: BBB model replicate 1


title: CD11C+ CD8T 2


title: stem cell-derived astrocytes replicate 3

source_name: brain astrocytes

value: Induced pluripotent stem cell-derived astrocytes


title: CD11C+ CD8T 3


title: stem cell-derived astrocytes replicate 1


title: S_92 Bulk RNA-seq

city: New York City

first: Yoko

last: Nomura

state: NY

address: 1470 Madison Ave

institute: Icahn School of Medicine at Mt. Sinai

data_processing:
Alignment and filtering with STAR
Annotation of exonic reads with featureCounts
Gene counts filtered and normalized in DESeq2 with variance stabilizing transformation
Genome_build: hg38
Supplementary_files_format_and_content: normalized gene counts

source_name: Bulk RNA-seq

extract_protocol:
Trizol
TruSeq

tag: tissue

value: placental tissue

tag: Sex

value: female

tag: maternal cannabis use

value: yes

tag: maternal cigarette smoking

value: no

tag: maternal age

tag: birth weight (g)

tag: gestational age (weeks)


title: stem cell-derived astrocytes replicate 2


title: S_91 Bulk RNA-seq

value: male


title: stem cell-derived endothelial cells replicate 1

source_name: brain endothelial cells

value: Induced pluripotent stem cell-derived brain endothelial cells


title: stem cell-derived endothelial cells replicate 3


title: S_99 Bulk RNA-seq


title: PC-3 Zeb1 KD DMSO 4

type: genomic

city: London

first: jenna

last: kitz

state: Ontario

address: 305 Central Ave, Unit 1

department: Anatomy and Cell Biology

country: Canada

institute: Western University

hyb_protocol: bisulphite converted DNA was amplified, fragmented and hybridised to Illumina Infinium Methylation EPIC Beadchip using standard Illumina protocol

scan_protocol: Arrays were imaged using iScan using standard recommended Illumina scanner setting

data_processing: Quality control was performed with the Chip Analysis Methylation Pipeline (ChAMP). This method filtered probes with a detection p-value above 0.01 (removing 3337 probes), bead count <3 in at least 5% of samples (removing 26519 probes). After probe filtering with ChAMP, sample were normalized with BMIQ normalization

label: Biotin-labled nucleotide

molecule: genomic DNA

label_protocol: Standard Illumina Protocol

growth_protocol: Human mesenchymal PC-3 prostate cancer cells (ATCC, Manassas, VA) were cultured in F12K media + 10% fetal bovine serum (FBS).

extract_protocol: DNA extraction was performed according to the Qiagen Blood and Cell Culture DNA Mini Kit protocol (Cat. #13323).

treatment_protocol: To create PC-3 Zeb1KD and ctrl cells, 1x106 PC-3cells/mL were seeded into each well of a 6-well dish 24 h prior to transduction. Twenty-five μl of SMARTvector Lentiviral Zeb1 shRNA stock (target region; 3’ untranslated region, target sequence 5’-TCTAAACCCAGGCTTCCCT-3’) or scrambled control (Dharmacon, Ottawa CA) was added to each well and growth media was exchanged for transduction media containing 0.01% Polybrene. Twenty hours after transduction, transduction media was exchanged for growth media. One day later, growth media was exchanged for selection media containing 0.025% puromycin. Cells were then cultured as usual, supplementing growth media with 0.025% puromycin to continue selective pressure. Resulting changes in inducible Zeb1 expression (± Dox) were analyzed using immunoblotting and qRT-PCR

tag: zeb1 knockdown

value: Zeb1 Knockdown

tag: treated or untreated

value: DMSO


title: BBB model replicate 3


title: S_98 Bulk RNA-seq


title: S_97 Bulk RNA-seq


title: S_88 Bulk RNA-seq


title: S_96 Bulk RNA-seq


title: S_93 Bulk RNA-seq


title: S_90 Bulk RNA-seq


title: PC-3 Control DMSO 4

value: Scrambled Control


title: PC-3 Zeb1 KD 5-azacytidine 4


title: PC-3 Control 5-azacytidine 3


title: PC-3 Zeb1 KD 5-azacytidine 3


title: S_83 Bulk RNA-seq


title: PC-3 Zeb1 KD 5-azacytidine 2


title: S_9 Bulk RNA-seq


title: S_86 Bulk RNA-seq


title: S_89 Bulk RNA-seq


title: PC-3 Control 5-azacytidine 4


title: S_85 Bulk RNA-seq


title: S_76 Bulk RNA-seq


title: S_73 Bulk RNA-seq


title: S_72 Bulk RNA-seq


title: S_70 Bulk RNA-seq


title: PC-3 Zeb1 KD DMSO 2


title: S_7 Bulk RNA-seq


title: S_69M Bulk RNA-seq


title: S_84 Bulk RNA-seq


title: PC-3 Control DMSO 2


title: S_81 Bulk RNA-seq


title: PC-3 Zeb1 KD 5-azacytidine 1


title: PC-3 Control 5-azacytidine 2


title: S_67 Bulk RNA-seq


title: PC-3 Zeb1 KD DMSO 3


title: stem cell-derived endothelial cells replicate 2


title: S_80 Bulk RNA-seq


title: S_8 Bulk RNA-seq


title: S_65 Bulk RNA-seq


title: S_75 Bulk RNA-seq


title: S_60 Bulk RNA-seq


title: S_64 Bulk RNA-seq


title: S_6 Bulk RNA-seq


title: S_56 Bulk RNA-seq


title: S_55 Bulk RNA-seq


title: S_50 Bulk RNA-seq


title: S_58 Bulk RNA-seq


title: S_53 Bulk RNA-seq


title: S_54 Bulk RNA-seq


title: PC-3 Control DMSO 3


title: S_51 Bulk RNA-seq


title: S_47 Bulk RNA-seq


title: S_48 Bulk RNA-seq


title: PC-3 Control 5-azacytidine 1


title: S_46 Bulk RNA-seq


title: PC-3 Control DMSO 1


title: S_49 Bulk RNA-seq


title: S_59 Bulk RNA-seq


title: S_47Q Bulk RNA-seq


title: S_44 Bulk RNA-seq


title: S_50Q Bulk RNA-seq


title: S_38 Bulk RNA-seq


title: S_37Q Bulk RNA-seq


title: S_363Q Bulk RNA-seq


title: S_42 Bulk RNA-seq


title: S_37 Bulk RNA-seq


title: S_35 Bulk RNA-seq


title: S_40 Bulk RNA-seq


title: PC-3 Zeb1 KD DMSO 1


title: S_4 Bulk RNA-seq


title: S_34 Bulk RNA-seq


title: S_33 Bulk RNA-seq


title: S_329Q Bulk RNA-seq


title: S_43 Bulk RNA-seq


title: S_36 Bulk RNA-seq


title: S_28 Bulk RNA-seq


title: S_41 Bulk RNA-seq


title: S_29 Bulk RNA-seq


title: S_77 Bulk RNA-seq


title: S_282M Bulk RNA-seq


title: S_275M Bulk RNA-seq


title: S_27 Bulk RNA-seq


title: S_268M Bulk RNA-seq


title: S_26 Bulk RNA-seq


title: S_252Q Bulk RNA-seq


title: S_25 Bulk RNA-seq


title: S_232Q Bulk RNA-seq


title: S_32 Bulk RNA-seq


title: S_24 Bulk RNA-seq


title: S_23 Bulk RNA-seq


title: S_311Q Bulk RNA-seq


title: S_238Q Bulk RNA-seq


title: S_30 Bulk RNA-seq


title: S_3 Bulk RNA-seq


title: S_243M Bulk RNA-seq


title: S_21 Bulk RNA-seq


title: S_20Q Bulk RNA-seq


title: S_204Q Bulk RNA-seq


title: S_28Q Bulk RNA-seq


title: S_18 Bulk RNA-seq


title: S_15 Bulk RNA-seq


title: S_31 Bulk RNA-seq


title: S_182Q Bulk RNA-seq


title: S_144 Bulk RNA-seq


title: S_19 Bulk RNA-seq


title: S_204M Bulk RNA-seq


title: S_16 Bulk RNA-seq


title: S_143 Bulk RNA-seq


title: S_22 Bulk RNA-seq


title: S_142 Bulk RNA-seq


title: S_140 Bulk RNA-seq


title: S_137 Bulk RNA-seq


title: S_136 Bulk RNA-seq


title: S_139 Bulk RNA-seq


title: S_132 Bulk RNA-seq


title: S_14 Bulk RNA-seq


title: S_125 Bulk RNA-seq


title: S_126 Bulk RNA-seq


title: S_130 Bulk RNA-seq


title: S_17 Bulk RNA-seq


title: S_128 Bulk RNA-seq


title: S_135 Bulk RNA-seq


title: S_124 Bulk RNA-seq


title: S_123 Bulk RNA-seq


title: S_12 Bulk RNA-seq


title: S_134 Bulk RNA-seq


title: S_118 Bulk RNA-seq


title: S_120 Bulk RNA-seq


title: S_114 Bulk RNA-seq


title: S_116 Bulk RNA-seq


title: S_111 Bulk RNA-seq


title: S_115 Bulk RNA-seq


title: S_122 Bulk RNA-seq


title: S_113 Bulk RNA-seq


title: S_10Q Bulk RNA-seq


title: S_11 Bulk RNA-seq


title: S_109 Bulk RNA-seq


title: S_106 Bulk RNA-seq


title: S_108 Bulk RNA-seq


title: Optic nerve S4

city: Freiburg

first: Clemens

last: Lange

address: Kilianstraße 5

department: Klinik für Augenheilkunde

institute: Uniklinik Freiburg

data_processing:
Quality control FastQC Galaxy Version 0.72
Reads were mapped to the human reference genome (hg38, gencode release 34) with RNA STAR Galaxy Version 2.7.2b (default parameters) using the Gencode annotation file (Gencode 34)
Reads mapped to the human reference genome were counted using featureCounts Galaxy Version 1.6.4 (default parameters) using the aforementioned annotation file.
The output of featureCounts was imported to RStudio (Version 1.2.1335, R Version 3.5.3). Gene symbols and gene types were determined based on ENSEMBL release 100 (Human genes, GRCh38.p12, download on 05/25/2020). Reads were normalized using the R package DESeq2 Version 1.22.2 24 with default parameters.
Genome_build: hg38

source_name: Optic nerve

extract_protocol:
Total RNA was isolated from ten to fifteen formalin-fixed and paraffin-embedded (FFPE) 4-µm-thick sections of all specimens using the Quick-RNA FFPE Kit (Zymo Research, USA). Following a DNAse I digestion using the Baseline-ZERO kit (Epicentre, USA), the RNA concentration was measured with the Qubit RNA HS Assay Kit on a Qubit Fluorometer (Life Technologies, USA). The RNA quality was determined with the RNA Pico Sensitivity Assay on a LabChip GXII Touch (PerkinElmer, USA). The fragment size of all RNA samples ranged between 120 and 150 bp.
RNA sequencing was performed using massive analysis of cDNA ends (MACE), a 3' RNA sequencing method. The preparation of MACE libraries was carried out using 1 µg of total RNA, as previously described. The barcoded libraries were sequenced simultaneously on the NextSeq 500 (Illumina, USA) with 1× 75 bp. PCR bias was removed using an unique molecular identifier.

value: Optic nerve

tag: disease state

value: healthy


title: S_107 Bulk RNA-seq


title: S_103 Bulk RNA-seq


title: Optic nerve S3


title: Lid S5

source_name: Eye lid

value: Eye lid


title: S_100 Bulk RNA-seq


title: S_10 Bulk RNA-seq


title: S_1 Bulk RNA-seq


title: Lid S2


title: LDL_Subject_19

city: Nashville

first: Ryan

middle: M

last: Allen

state: TN

address: 2220 Pierce Ave

department: Medicine

institute: Vanderbilt University Medical Center

description: After adapter drimming with Cutadapt, identical reads were consolidated and counted

data_processing:
Trimmed to remove adapter sequences with Cutadaptv1.16
Aligned to hg19, human rRNA and tRNA databases using Bowtie1 v1.1.2
Reads that failed to align to host were aligned to exogenous RNA databases/genomes using the TIGER informatics pipeline.
Genome_build: hg19
Supplementary_files_format_and_content: Geo_Allen_AllTaskReads_Summary.csv indicates the total number of reads aligned to individual segments of the TIGER pipeline, and the number of reads that failed to align
Supplementary_files_format_and_content: Geo_Allen_NonParallel_TaskReadsSummary.csv indicates the total number of reads that aligned to the host genome and annotated host small RNA, reads that were too short for alignment, total reads that were aligned to non-host databases after removing redundant mapping, and reads that were not able to be aligned.
Supplementary_files_format_and_content: Geo_Allen_LDLsRNA_Alignment.xlsx contains the main mapping results to individual segments of the TIGER pipeline.

source_name: nLDL

extract_protocol:
Total RNA from density-isolated nLDL was performed using Qiagen miRNeasy RNA isolation kits.
Libraries were constructed using the NextFlex Small RNA Library Preparation kits V3 (Perkin-Elmer), size-selected (136-200 bp products) using PippinPrep (SageSciences)

tag: subject #


title: Optic nerve S2


title: LDL_Subject_16


title: LDL_Subject_17


title: LDL_Subject_18


title: Optic nerve S1


title: Lid S6


title: LDL_Subject_14


title: Lid S4


title: Lid S1


title: Lid S3


title: LDL_Subject_20


title: LDL_Subject_15


title: LDL_Subject_12


title: LDL_Subject_9


title: LDL_Subject_10


title: LDL_Subject_11


title: LDL_Subject_8


title: LDL_Subject_6


title: LDL_Subject_7


title: LDL_Subject_1


title: S_262Q Bulk RNA-seq


title: LDL_Subject_2


title: LDL_Subject_5


title: Lept23

city: Cambridge

first: Felix

last: Krueger

address: Granta Park

department: Bioinformatics

country: United Kingdom

institute: Altos Labs

description: Breeding pairs of C57BL/6J (B6) mice were obtained from the Jacksons Laboratory (The Jacksons Laboratory, Bar Harbor, Maine, USA). At 8 weeks (wk) of age B6 animals were divided into 2 groups (n=10/group). In DIO, the control group was fed ad libitum chow diet (CD, 11% energy in kcal from fat, 5053, rodent diet 20, LabDiet IPS, London, UK), while the experimental group received high fat diet (HFD, 58% energy in kcal from fat, AIN-76A 9G03, LabDiet IPS). Mice were maintained under the diet for 4 or 16 wk. Regarding the pharmacological hyperleptinemia protocol (LEPT), 8 wk old B6 female mice were divided into 4 groups (n=15/group): i) saline 9 days (d); ii) leptin 9 d; iii) saline 16 d; and iv) leptin 16 d (Recombinant Mouse Leptin, GFM26, Cell Guidance Systems, Cambridge, UK). The animals were injected intraperitoneally twice a day, at 09:00h and 21:00h and total dosage of 100g/day of leptin was administrated. For all protocols, mice were housed with a 12h light/12h dark cycle at room temperature. A total of approximately 50 cumulus cells were collected from one individual cumulus oophorous complex, obtained after superovulation of female mice from either 4 wk and 16 wk DIO or 16 d leptin protocols.

data_processing:
Trim galore v0.4.2 was used with default parameters on raw fastq sequence files.
Mapping of the RNA-seq data was done with HISAT2 v2.0.5 against the mouse GRCm38 genome, as guided by known splice sites taken from Ensembl v68.
Genome_build: GRCm38
Matrix (.txt) of raw counts

organism: Mus musculus

source_name: Lept

extract_protocol:
Cumulus cells were collected into RLT buffer (1053393, Qiagen, Hilden, Germany) and kept in -80oC until library generation. mRNA was captured using Smart-seq2 oligo-dT pre-annealed to magnetic beads (MyOne C1, Invitrogen, Carlsbad, California, USA).
The beads were resuspended in 10 μl of reverse transcriptase mix (100 U, SuperScript II, Invitrogen; 10 U, RNAsin, Promega, Madison, Wisconsin, USA), 1 × Superscript II First-Strand Buffer, 2.5 mM ditiotreitol (DTT, Invitrogen), 1 M betaine (Sigma Aldrich), 9 mM magnesium chloride (MgCl2, Invitrogen), 1 μM Template-Switching Oligo (Exiqon, Vedbaek, Denmark), 1 mM dNTP mix (Roche) and incubated for 60 min at 42 °C followed by 30 min at 50 °C and 10 min at 60 °C (Picelli et al., 2013, 2014). Amplification of the cDNA was then undertaken after adding 11 μl of 2 × KAPA HiFi HotStart ReadyMix and 1 μl of 2 μM ISPCR primer (Picelli et al., 2013, 2014), followed by the cycle: 98 °C for 3 min, then 9 cycles of 98 °C for 15 s, 67 °C for 20 s, 72 °C for 6 min and finally 72 °C for 5 min. Finally, the cDNA was purified using similar volume of AMPure beads (Beckman Coulter, Brea, California, USA) and eluted into 20 μl of water. All libraries were prepared from 100 to 200 pg of cDNA using the Nextera XT Kit (Illumina, San Diego, California, USA), regarding the manufacturer’s instructions. The final cDNA library was purified using a 0.7:1 volumetric ratio of AMPure beads before pooling and sequencing.

value: cumulus cells

tag: group

value: leptin treated


title: Lept21


title: S_2 Bulk RNA-seq


title: Lept18


title: Lept20


title: Cont_Lept3

source_name: Cont Lept3

value: leptin control


title: Cont_Lept2

source_name: Cont Lept2


title: LDL_Subject_13


title: SP CD8 WT Chip input

city: Shanghai

first: zheyi

last: chen

address: 1665 Kong Jiang Road

institute: Shanghai Jiao Tong University School of Medicine

library_source: genomic

data_processing:
For ChIP-Seq, raw reads were filtered to obtain high-quality clean reads by removing sequencing adapters, short reads (length <35 bp) and low-quality reads using Cutadapt (v1.9.1) and Trimmomatic (v0.35). The clean reads were mapped to the mouse genome (assembly GRCm38) using Bowtie2 (v2.2.6) software. Peak detection was performed using the MACS (v2.1.1) peak finding algorithm with 0.01 set as the p-value cutoff. Annotation of peak sites to gene features was performed using the ChIPseeker R package. Genome graphs were generated and viewed using IGV (Integrative Genomics Viewer), and we aligned the H4R3me2s, HR8me2s and input control in CD8+ T cells.
Genome_build: GRCm38
Supplementary_files_format_and_content: For ChIP-Seq, bigWig file contain Chromosome segment information.

source_name: Spleen

extract_protocol:
Single-cell suspensions were harvested from fresh tissues according to standard procedures and surface-stained in FACS buffer with monoclonal antibodies. Related T cell subsets from mice spleen or thymus were sorted with FACS Aria II Cell Sorter.  For Chip-seq, after the cells were crosslinked and sonicated, the fragmented chromatin fragments were precleared and then immunoprecipitated with Protein A + G magnetic beads coupled with antibodies.
For Chip-seq, after reverse crosslinking, ChIP and input DNA fragments were end-repaired and a-tailed using the NEBNext End Repair/dA-Tailing Module (E7442, NEB) followed by adaptor ligation with the NEBNext Ultra Ligation Module (E7445, NEB). The DNA libraries were amplified for 15 cycles and sequenced using Illumina NextSeq 500 in single-end 1x75 sequencing mode.

tag: strain

value: T cells

tag: chip antibody

tag: genotype

value: wild type


title: SP CD8 WT Chip H3R8me2s

tag: cell type

value: H3R8me2s (Epigentek cat#A-3706)


title: SP CD8 WT Chip H4R3me2s

value: H4R3me2s (Active Motif cat#61187)


title: SP CD8 Prmt5-CKO Chip H4R3me2s

value: CD4-Cre+ PRMT5fl/fl


title: SP CD8 Prmt5-CKO Chip input


title: 4wk_HFD3

source_name: 4wk HFD3

value: high-fat diet 4 weeks


title: S_265M Bulk RNA-seq

value: placental tissue

value: male

value: no


title: 4wk_HFD14

source_name: 4wk HFD14


title: Cont_Lept1

source_name: Cont Lept1


title: LDL_Subject_3


title: 4wk_HFD5

source_name: 4wk HFD5


title: 4wk_CD11

source_name: 4wk CD11

value: chow diet 4 weeks


title: 4wk_CD7

source_name: 4wk CD7


title: 4wk_CD1

source_name: 4wk CD1


title: LDL_Subject_4


title: 4wk_HFD13

source_name: 4wk HFD13


title: SP CD8 Prmt5-CKO Chip H3R8me2s


title: 16wk_HFD_lowg21

source_name: 16wk HFD lowg21

value: high-fat diet 16 weeks low weight gainer


title: 16wk_HFD_lowg23

source_name: 16wk HFD lowg23


title: 4wk_CD8

source_name: 4wk CD8


title: 16wk_HFD6

source_name: 16wk HFD6

value: high-fat diet 16 weeks


title: 4wk_HFD4

source_name: 4wk HFD4


title: 16wk_HFD11

source_name: 16wk HFD11


title: 16wk_HFD12

source_name: 16wk HFD12


title: Lept19

source_name: Lept


title: 16wk_HFD16

source_name: 16wk HFD16


title: 16wk_HFD15

source_name: 16wk HFD15


title: 4wk_CD2

source_name: 4wk CD2


title: 16wk_CD13

source_name: 16wk CD13

value: chow diet 16 weeks


title: Murine macrophage - PDL1_Neg_rep2

city: East Lansing

first: Andrew

last: Olive

state: MI

address: 567 Wilson Road

department: Microbiology and Molecular Genetics

institute: Michigan State University

description:
Replicate 2
PDL1_neg2

data_processing:
Library strategy: Amplicon sequencing of sgRNA loci (CRISPR screen)
Illumina software was used for Basecalls
Cutadapt (v2.9) was used to remove primer adapter sequences (i.e. vector sequence flanking the sgRNA)
sgRNA index was prepared comprising sgRNAs that target genes which are transcribed in macrophage cell line (Kiritsy et al., 2020a)
Bowtie2 via MAGeCK-MLE was used to map trimmed sequencing reads to sgRNA index (no mismatch allowed) and to generate a read/count file for each library
MAGeCK-MLE was used to carry out subsequent analyses to account for variable sequencing depth and to facilitate multiple conditon-specific comparisons
Genome_build: mm10
Supplementary_files_format_and_content: Matrix table with raw and normalized sgRNA abundances for each sample

source_name: Macrophage cell line (L3 clone)

growth_protocol: Macrophage cell line was established by immortalization of bone marrow from C57BL/6J male mouse using J2 virus while cultured in complete DMEM+10%FBS enriched with L929-supernatants. L929 conditioned media was weaned to establish growth-factor independence. Cell line was transduced with a lentivirus expressing Cas9 that conferred hygromycin resistance. Two days after transduction, cells were selected with hygromycin at 50ug/mL until non-transduced control cells were >99% dead. Individual clones were isolated by serial dilution and clones were screened for Cas9 expression by immnoblot and functionally by gene editing. The L3 clone was selected for subsequent use with a genome-wide library of sgRNAs (Brie). Following geneation of the lentiviral library and titering of the viral supernatants, L3 cells were transduced at an MOI of ~0.2 and after 48h, cells were selected with puromycin (2.5ug/mL) before aliquots of the selected library were frozen. For screens, aliquot were thawed and minimally expanded to avoid skewing of library representation before treatment and selection.

extract_protocol:
Fixed sortd cell populations underwent crosslink reversal before genomic DNA isolation using DNEasy (Qiagen) according the the manufacterer's instructions.
Sequencing libraries were generated using previously published instructions and primers for the Brie library (Doench et al., 2016; Joung et al., 2017). Briefly, the sgRNA loci was PCR amplified using custom primers compatibile with Illumina sequencing that facilitated sample multiplexing with reverse primer indices. Following amplification of indivudal libraries with a unique index, samples were pooled and sequenced on an Illumina NextSeq 500 (either 75 or 150 cycle)

treatment_protocol: Recombinant murine interferon gamma (10ng/mL) for 24h. Cells were scraped to collect and stained with a viability dye and the indicated fluorescent antibody at the optimized concentrations. Following staining, cells were fixed in 4% paraformaldehyde and sorted by FACS to isolate high and low expressing populations.

value: Bone marrow

tag: derivation

value: Macrophage, J2 virus immortalized

tag: crispr library

value: Brie (Doenh et al., 2016) KO library:Lentiviral Cas9-Hyg + genome-wide library of lentiviral sgRNA-Puro

tag: treatment, selection

value: IFNg (24h), PDL1 low

tag: facs antibody

value: APC anti-mouse CD274 (B7-H1, PD-L1) Antibody, Clone 10F.9G2 Biolegend 124311

tag: sequencing primers reference

value: Joung et al., 2017


title: 16wk_HFD10

source_name: 16wk HFD10

value: cumulus cells


title: 16wk_CD9

source_name: 16wk CD9


title: 16wk_CD5

source_name: 16wk CD5


title: 16wk_CD2

source_name: 16wk CD2


title: Murine macrophage - PDL1_Pos_rep2

description:
Replicate 2
PDL1_pos2

tag: tissue

value: IFNg (24h), PDL1 high


title: Murine macrophage - PDL1_Input_rep1

description:
Replicate 1
PDL1_input1

value: IFNg (24h)


title: Murine macrophage - PDL1_Pos_rep1

description:
Replicate 1
PDL1_pos1


title: Murine macrophage - PDL1_Neg_rep1

description:
Replicate 1
PDL1_neg1


title: 16wk_HFD_lowg22

source_name: 16wk HFD lowg22


title: Murine macrophage - CD40_Input_rep2

description:
Replicate 2
CD40_input2

value: APC anti-mouse CD40 Antibody, Clone 3/23 Biolegend 124611

value: Doench et al. 2016


title: Murine macrophage - CD40_Pos_rep2

description:
Replicate 2
CD40_pos2

value: IFNg (24h), CD40 high


title: Murine macrophage - MHCII_Neg_rep1

description:
Replicate 1
MHC_neg1

value: IFNg (24h), MHCII low

value: APC anti-mouse I-A/I-E Antibody, Clone M5/114.15.2 Biolegend 107613


title: Murine macrophage - MHCII_Input_rep2

description:
Replicate 2
MHC_input2


title: Murine macrophage - CD40_Neg_rep1

description:
Replicate 1
CD40_neg1

value: IFNg (24h), CD40 low


title: 16wk_CD1

source_name: 16wk CD1


title: Murine macrophage - MHCII_Neg_rep2

description:
Replicate 2
MHC_neg2


title: 16wk_HFD14

source_name: 16wk HFD14

value: high-fat diet 16 weeks


title: Murine macrophage - CD40_Input_rep1

description:
Replicate 1
CD40_input1


title: intestine WT rep4

city: Ithaca

first: Natasza

last: Kurpios

address: Vet Medical Center, 930 Campus Road

institute: Cornell University

description: WT 335

data_processing:
Illumina pipeline software v1.8 was used for base calling.
trim_galore was used to trim and filter reads (--nextseq 20 --length 50)
STAR was used to map reads to the Mus musculus mm10 reference using Ensembl gene annotations and output raw counts (--quantMode GeneCounts)
Genome_build: mm10

source_name: small intestine

extract_protocol:
Total RNA was isolated with Trizol, with an extra chloroform extraction to remove residual phenol and addition of glyco-blue as a carrier to promote RNA precipitation. RNA concentration was determined on a Nanodrop.
TruSeq-barcoded RNAseq libraries were generated with the NEBNext Ultra II Directional RNA Library Prep Kit (New England Biolabs) with polyA+ enrichment using 1 ug total RNA as input.

value: mix background

value: intestine

tag: age

value: postnatal day 1.5

value: WT


title: intestine mut rep1

description: ASE 302

value: ASE-/-


title: Murine macrophage - CD40_Neg_rep2

description:
Replicate 2
CD40_neg2

value: Bone marrow

value: Macrophage, J2 virus immortalized

value: Brie (Doenh et al., 2016) KO library:Lentiviral Cas9-Hyg + genome-wide library of lentiviral sgRNA-Puro


title: 16wk_CD8

source_name: 16wk CD8

value: cumulus cells

value: chow diet 16 weeks


title: Murine macrophage - MHCII_Pos_rep1

description:
Replicate 1
MHC_pos1

value: IFNg (24h), MHCII high


title: Murine macrophage - MHCII_Pos_rep2

description:
Replicate 2
MHC_pos2

value: Doench et al. 2016


title: Murine macrophage - CD40_Pos_rep1

description:
Replicate 1
CD40_pos1

value: IFNg (24h), CD40 high

value: APC anti-mouse CD40 Antibody, Clone 3/23 Biolegend 124611


title: Murine macrophage - MHCII_Input_rep1

description:
Replicate 1
MHC_input1

value: IFNg (24h)


title: TZPL47_Placenta of wild type 6-somite embryo (Jx1 cross) #3

city: Grand Rapids

first: Piroska

middle: E

last: Szabó

address: 333 Bostwick ave NE

department: Epigenetics

institute: Van Andel Institute

description:
WT.SIX.J1.3
ZYG_WT_J1_6SOM_M_PL61

data_processing:
Sequencing Reads were aligned aligned to the mm10 genome using STAR(v2.6.0c) with parameters -outFilterType BySJout --outFilterMultimapNmax 20 --alignSJoverhangMin 8 --alignSJDBoverhangMin 1 --alignIntronMax 1000000 --alignMatesGapMax 1000000 --outFilterMismatchNmax 999 --twopassMode Basic --chimSegmentMin 20 --alignIntronMin 20. Counts per gene for each sample were calculated using featureCounts(v1.6.1) with parameters -P  -s 1 --primary -Q 20 –ignoreDup.
For allele-specific analysis, reads were aligned to a custom 129SvImJ-Jf1/MsJ SNP masked genome using hisat2(v2.1.0) with parameters –sp 1000,1000 –dta-cufflinks. To seperate reads mapping to the 129SvImJ or JF1MsJ genome, we used SNPsplit(v0.3.2) with paired-end mode.
Both total RNA-seq and allele-specific were generated using deeptools(v3.2.1) bamCoverage with parameters –normalizeUsing CPM –effectiveGenomeSize 2652783500 -bs 10
For transposable element analysis, uniquely mapped reads were extracted from the STAR aligned bam file using samtools(v1.9) with parameters -q 255. Using the repeatmasker mm10 annotation file, uniquely mapped counts per sample were determined using featureCounts(v1.6.1) with parameters -s1 –primary -Q20 –ignoreDup.
Genome_build: mm10
Supplementary_files_format_and_content: Matrix Table with raw gene counts for every genes and every sample
Supplementary_files_format_and_content: Matrix Table with raw allele gene counts for every genes and every sample
Supplementary_files_format_and_content: Matrix Table with raw repeat counts for every genes and every sample
Supplementary_files_format_and_content: Bigwigs that contain total RNA-seq CPM expression values
Supplementary_files_format_and_content: Bigwigs that contain allele specific CPM expression values

extract_protocol:
Total RNA was isolated from placentas by acid-phenol extraction using RNA-Bee (Tel-TEST) followed by isopropanol precipitation. Genomic DNA was removed with the DNA-free Kit (Ambion).
Total RNA ibraries were prepared by the Van Andel Genomics Core from 500 ng of total RNA using the KAPA RNA HyperPrep Kit with RiboseErase (v1.16) (Kapa Biosystems, Wilmington, MA USA) following kit parameters. In short, RNA was sheared for 6 min at 86 oC to produce 300-400 bp fragments and it was converted to cDNA. After repairing the ends the cDNA fragments were ligated to Bioo Scientific NEXTflex Adapters (Bioo Scientific, Austin, TX, USA) and amplified using 11 cycles of PCR. Quality and quantity of the finished libraries were assessed using a combination of Agilent DNA High Sensitivity chip (Agilent Technologies, Inc.), QuantiFluor® dsDNA System (Promega Corp., Madison, WI, USA), and Kapa Illumina Library Quantification qPCR assays (Kapa Biosystems).  Individually indexed libraries were pooled, and paired end 75 bp sequencing was performed on an Illumina NextSeq 500 sequencer using a 150 bp HO sequencing kit (v2) (Illumina Inc., San Diego, CA, USA), with all libraries run across 5 flowcells to return a minimum read depth of 40M read pairs per library. Base calling was done by Illumina NextSeq Control Software (NCS) v2.0 and output of NCS was demultiplexed and converted to FastQ format with Illumina Bcl2fastq v1.9.0.
Allele-specific, stranded, paired-end total RNA seq

treatment_protocol: An Ehmt2 conditional knockout mouse line (Ehmt2fl/fl) was generated in our laboratory by gene targeting in 129S1/SvImJ ES cells (Zeng, Han et al. PNAS 2019). Ehmt2fl/fl and Ehmt2+/−,129S mice were maintained on 129S1 background. The floxed SET domain was removed to generate the Ehmt2−(129S1) allele by crossing the Ehmt2fl/+ male with 129S1/Sv-Hprttm1(CAGcre)Mann/J transgenic mouse female (Tang, Silva et al. 2002). Placentas were dissected between 8.5 dpc and 9.5 dpc, and were genotyped for Ehmt2 mutation status and for sex by PCR using their allantois as described (Zeng, Han et al. 2019). Ehmt2−/− homozygous and Ehmt2+/+ wild type placentas were obtained by natural mating of Ehmt2+/− females and males. To distinguish the paternal and maternal alleles, we used F1 hybrid samples collected from reciprocally crossedparents from the inbred mouse strains 129S1 (Mus musculus musculus) and JF1/Ms (Mus musculus molossinus) (JF1). We backcrossed the Ehmt2fl(129S1) allele to the JF1 mouse strain for more than ten generations to obtaine the Ehmt2−(JF1.N10) allele, where the genome (except the Ehmt2 locus and its close neighbors, in the approximate interval of chr 17:34647146-35241746) has been replaced by JF1 chromosomes during meiotic recombination events. Crossing a female carrying this allele with the original Ehmt2−(129S1) male yielded the Ehmt2−(JF1.N10)/−(129S1) zygotic mutant and Ehmt2+(JF1)/+(129S1) wild type placentas. The reciprocal cross resulted in Ehmt2−(129S1)/−(JF1.N10) mutant and Ehmt2+(129S1)/+(JF1) wild type placentas. Control Ehmt2+(129S1)/+(JF1) placentas were obtained from wild typel JF1 and 129S1 mice. Ehmt2fl/fl; Zp3-CreTg/0 females were crossed with wild type JF1 males to generate Ehmt2mat−/+JF1 placentas. Their control came from crossing Ehmt2fl/fl; Zp3-CreTg- females with wild type JF1 males. Ehmt2fl/fl; Zp3-CreTg/0 females were crossed with Ehmt2+/−(JF1.N10) males to generate Ehmt2mat−/zyg-JF1 placentas. Ehmt2+/- (JF1.N10) dams and wild-type 129S1 sires yielded maternal haploinsufficient wild type placentas. Ehmt2+/-(129S1) sire and JF1 dam yielded paternal haploinsufficient wild type placentas.

tag: sample type

value: WILD TYPE

tag: embryo genotype

value: wild type

tag: genotype of maternal germ line

value: heterozygous mutant

tag: genotype of father

tag: somite number

value: SIX

tag: cross

value: JF1/MsJ x 129SvImJ

tag: cross abbreviation

tag: Sex

value: male

tag: age (dpc) adjusted to cross

tag: day of collection (gestational day)

tag: time of collection


title: intestine mut rep3

description: ASE 349

tag: strain

value: mix background

value: intestine

value: postnatal day 1.5

tag: genotype

value: ASE-/-


title: intestine WT rep2

description: WT 300

value: WT


title: intestine WT rep3

description: WT 303


title: intestine WT rep1

description: WT 322


title: S_297Q Bulk RNA-seq

source_name: Bulk RNA-seq

value: placental tissue

value: female

tag: maternal cannabis use

value: no

tag: maternal cigarette smoking

tag: maternal age

tag: birth weight (g)

tag: gestational age (weeks)


title: TZPL44_Placenta of wild type 12-somite embryo (Jx1 cross) #4

description:
WT.TWELVE.J1.4
ZYG_WT_J1_12SOM_M_PL72Dec

tag: sample type

tag: embryo genotype

value: TWELVE


title: TZPL45_Placenta of wild type 6-somite embryo (Jx1 cross) #1

description:
WT.SIX.J1.1
ZYG_WT_J1_6SOM_F_PL71

tag: genotype of maternal germ line

tag: genotype of father


title: TZPL48_Placenta of wild type 6-somite embryo (Jx1 cross) #4

description:
WT.SIX.J1.4
ZYG_WT_J1_6SOM_M_PL72

tag: somite number

tag: cross


title: TZPL46_Placenta of wild type 6-somite embryo (Jx1 cross) #2

description:
WT.SIX.J1.2
ZYG_WT_J1_6SOM_F_PL89

tag: cross abbreviation

tag: Sex


title: intestine mut rep2

description: ASE 305

tag: tissue

tag: age


title: TZPL43_Placenta of wild type 12-somite embryo (Jx1 cross) #3

description:
WT.TWELVE.J1.3
ZYG_WT_J1_12SOM_M_PL69-Dec

tag: age (dpc) adjusted to cross

tag: day of collection (gestational day)

tag: time of collection


title: TZPL42R_Placenta of wild type 12-somite embryo (Jx1 cross) #2

description:
WT.TWELVE.J1.2
ZYG_WT_J1_12SOM_F_PL102


title: TZPL38R_Placenta of wild type 6-somite embryo (1xJ cross) #2

description:
WT.SIX.1J.2
ZYG_WT_1J_6SOM_F_PL75

value: 129SvImJ x JF1/MsJ


title: TZPL39_Placenta of wild type 6-somite embryo (1xJ cross) #3

description:
WT.SIX.1J.3
ZYG_WT_1J_6SOM_M_PL22


title: TZPL40_Placenta of wild type 6-somite embryo (1xJ cross) #4

description:
WT.SIX.1J.4
ZYG_WT_1J_6SOM_M_PL39


title: TZPL41_Placenta of wild type 12-somite embryo (Jx1 cross) #1

description:
WT.TWELVE.J1.1
ZYG_WT_J1_12SOM_F_PL38


title: TZPL37R_Placenta of wild type 6-somite embryo (1xJ cross) #1

description:
WT.SIX.1J.1
ZYG_WT_1J_6SOM_F_PL44


title: TZPL36_Placenta of homozygous Ehmt2 mutant 6-somite embryo (Jx1 cross) #4

description:
HOMO.SIX.J1.4
ZYG_HOMO_J1_6SOM_M_PL85

value: ZYGOTIC HOMOZYGOUS MUTANT

value: homozygous mutant


title: TZPL35_Placenta of homozygous Ehmt2 mutant 6-somite embryo (Jx1 cross) #3

description:
HOMO.SIX.J1.3
ZYG_HOMO_J1_6SOM_M_PL35


title: TZPL34_Placenta of homozygous Ehmt2 mutant 6-somite embryo (Jx1 cross) #2

description:
HOMO.SIX.J1.2
ZYG_HOMO_J1_6SOM_F_PL68Dec


title: TZPL31_Placenta of homozygous Ehmt2 mutant 12-somite embryo (Jx1 cross) #3

description:
HOMO.TWELVE.J1.3
ZYG_HOMO_J1_12SOM_M_PL40


title: TZPL30_Placenta of homozygous Ehmt2 mutant 12-somite embryo (Jx1 cross) #2

description:
HOMO.TWELVE.J1.2
ZYG_HOMO_J1_12SOM_F_PL48


title: TZPL29_Placenta of homozygous Ehmt2 mutant 12-somite embryo (Jx1 cross) #1

description:
HOMO.TWELVE.J1.1
ZYG_HOMO_J1_12SOM_F_PL47


title: TZPL32_Placenta of homozygous Ehmt2 mutant 12-somite embryo (Jx1 cross) #4

description:
HOMO.TWELVE.J1.4
ZYG_HOMO_J1_12SOM_M_PL41


title: TZPL28_Placenta of homozygous Ehmt2 mutant 6-somite embryo (1XJ cross) #4

description:
HOMO.SIX.1J.4
ZYG_HOMO_1J_6SOM_M_PL82


title: TZPL27_Placenta of homozygous Ehmt2 mutant 6-somite embryo (1XJ cross) #3

description:
HOMO.SIX.1J.3
ZYG_HOMO_1J_6SOM_M_PL59


title: TZPL26R_Placenta of homozygous Ehmt2 mutant 6-somite embryo (1XJ cross) #2

description:
HOMO.SIX.1J.2
ZYG_HOMO_1J_6SOM_F_PL132


title: TZPL24_Placenta of maternal Ehmt2 haploinsufficient 6-somite embryo (Jx1 cross) #4

description:
MATHAPLO_WT.SIX.J1.4
MATHAPLO_WT_J1_6SOM_M_PL21

value: MATERNAL HAPLOINSUFFICIENT WILD TYPE

value: wild type


title: TZPL25R_Placenta of homozygous Ehmt2 mutant 6-somite embryo (1XJ cross) #1

description:
HOMO.SIX.1J.1
ZYG_HOMO_1J_6SOM_F_PL117

value: heterozygous mutant

value: SIX


title: TZPL23_Placenta of maternal Ehmt2 haploinsufficient 6-somite embryo (Jx1 cross) #3

description:
MATHAPLO_WT.SIX.J1.3
MATHAPLO_WT_J1_6SOM_M_PL20

value: JF1/MsJ x 129SvImJ

value: male


title: TZPL21_Placenta of maternal Ehmt2 haploinsufficient 6-somite embryo (Jx1 cross) #1

description:
MATHAPLO_WT.SIX.J1.1
MATHAPLO_WT_J1_6SOM_F_PL18


title: TZPL20_Placenta of paternal Ehmt2 haploinsufficient 6-somite embryo (Jx1 cross) #4

description:
PATHAPLO_WT.SIX.J1.4
PATHAPLO_WT_J1_6SOM_M_PL30

value: PATERNAL HAPLOINSUFFICIENT WILD TYPE


title: TZPL22_Placenta of maternal Ehmt2 haploinsufficient 6-somite embryo (Jx1 cross) #2

description:
MATHAPLO_WT.SIX.J1.2
MATHAPLO_WT_J1_6SOM_F_PL24


title: TZPL19_Placenta of paternal Ehmt2 haploinsufficient 6-somite embryo (Jx1 cross) #3

description:
PATHAPLO_WT.SIX.J1.3
PATHAPLO_WT_J1_6SOM_M_PL29


title: b120: deplete_L_T4 B

city: Vancouver

first: Anwar

last: Alqattan

state: BC

address: 4200-6270 University Boulevard

department: Earth Ocean and Atmospheric Sciences/Oceanography

institute: University of British Columbia

data_processing:
Trimmomatic version 0.36 was used to clip NEB adapter sequences, trim for minimum trailing quality of 30 and filter reads for minimum length of 32.
RSEM version 1.3.1 was used to quantify genes as defined on ASM309565v1
Genome_build: ASM309565v1
Supplementary_files_format_and_content: File containing RSEM gene-level expression estimates. Details at http://deweylab.biostat.wisc.edu/rsem/rsem-calculate-expression.html#output

organism: Dokdonia sp. Dokd-P16

source_name: Bacterial culture

extract_protocol:
Total RNA from filtered cultured samples was extracted using RNeasy PowerWater kit (Qiagen, Hilden, Germany) following the manufacturer's instructions. The integrity of RNA samples was assessed using Bio-analyzer 2100 with the Agilent RNA 6000 Pico Kit (Agilent Technologies). The concentration of RNA samples was measured using the Qubit HS RNA kit on the Qubit 4. Extracted total RNA samples were sent to genome Quebec, Canada (https://cesgq.com/home) for rRNA depletion, library preparation and sequencing.
For RNA-seq analyses, rRNA was removed by Ribo-Zero rRNA Removal kit (Illumina). After depletion of rRNA, RNA-seq libraries were generated with NEBNext UltraII directional RNA library prep kit for Illumina (New England Biolabs) following the manufacturer’s instructions. The libraries were sequenced with Illumina Novaseq to generate paired-ends 100 bp reads.

tag: iron bioavailability

value: deplete

tag: media

value: L

tag: exposure time


title: b118: deplete_NOADD_Tend E

value: NOADD

value: Tend


title: TZPL33_Placenta of homozygous Ehmt2 mutant 6-somite embryo (Jx1 cross) #1

description:
HOMO.SIX.J1.1
ZYG_HOMO_J1_6SOM_F_PL65-2

value: female


title: TZPL18_Placenta of paternal Ehmt2 haploinsufficient 6-somite embryo (Jx1 cross) #2

description:
PATHAPLO_WT.SIX.J1.2
PATHAPLO_WT_J1_6SOM_F_PL34

value: WT


title: b117: deplete_L_T4 A


title: TZPL16_Placenta of maternal Ehmt2 mutant 6-somite embryo (1xJ cross) #4

description:
MATWT.SIX.1J.4
MAT_WT_1J_6SOM_M_PL7

value: MATERNAL CONTROL WILD TYPE

value: WT (fl/fl;Cre-)


title: TZPL17_Placenta of paternal Ehmt2 haploinsufficient 6-somite embryo (Jx1 cross) #1

description:
PATHAPLO_WT.SIX.J1.1
PATHAPLO_WT_J1_6SOM_F_PL26


title: b116: deplete_L_T1 C


title: TZPL12_Placenta of maternal Ehmt2 mutant 6-somite embryo (1xJ cross) #4

description:
MATMUT.SIX.1J.4
MAT_HET_1J_6SOM_M_PL50

value: MATERNAL MUTANT

value: homozygous mutant (fl/fl;Cre+)

value: 129SvImJ x JF1/MsJ


title: TZPL11_Placenta of maternal Ehmt2 mutant 6-somite embryo (1xJ cross) #3

description:
MATMUT.SIX.1J.3
MAT_HET_1J_6SOM_M_PL44

value: wild type


title: TZPL13_Placenta of maternal Ehmt2 mutant 6-somite embryo (1xJ cross) #1

description:
MATWT.SIX.1J.1
MAT_WT_1J_6SOM_F_PL5


title: TZPL14_Placenta of maternal Ehmt2 mutant 6-somite embryo (1xJ cross) #2

description:
MATWT.SIX.1J.2
MAT_WT_1J_6SOM_F_PL8


title: TZPL15_Placenta of maternal Ehmt2 mutant 6-somite embryo (1xJ cross) #3

description:
MATWT.SIX.1J.3
MAT_WT_1J_6SOM_M_PL6


title: b114: deplete_L_T3 E


title: TZPL10_Placenta of maternal Ehmt2 mutant 6-somite embryo (1xJ cross) #2

description:
MATMUT.SIX.1J.2
MAT_HET_1J_6SOM_F_PL48

value: heterozygous mutant


title: b113: deplete_AQUIL_T2 E

value: AQUIL


title: TZPL08_Placenta of control 6-somite embryo  (Jx1 cross) #4

description:
CONTROL.SIX.J1.4
CONTROL_WT_J1_6SOM_M_PL23

value: CONTROL

value: SIX

value: JF1/MsJ x 129SvImJ


title: TZPL07_Placenta of control 6-somite embryo  (Jx1 cross) #3

description:
CONTROL.SIX.J1.3
CONTROL_WT_J1_6SOM_M_PL20

value: male


title: TZPL09_Placenta of maternal Ehmt2 mutant 6-somite embryo (1xJ cross) #1

description:
MATMUT.SIX.1J.1
MAT_HET_1J_6SOM_F_PL10


title: b115: deplete_L_T0 B

value: deplete

value: L


title: TZPL06_Placenta of control 6-somite embryo  (Jx1 cross) #2

description:
CONTROL.SIX.J1.2
CONTROL_WT_J1_6SOM_F_PL24


title: b119: deplete_L_T0 D


title: TZPL04_Placenta of control 6-somite embryo  (1xJ cross) #4

description:
CONTROL.SIX.1J.4
CONTROL_WT_1J_6SOM_M_PL6


title: b112: deplete_AQUIL_T4 B


title: TZPL05_Placenta of control 6-somite embryo  (Jx1 cross) #1

description:
CONTROL.SIX.J1.1
CONTROL_WT_J1_6SOM_F_PL19


title: TZPL02_Placenta of control 6-somite embryo  (1xJ cross) #2

description:
CONTROL.SIX.1J.2
CONTROL_WT_1J_6SOM_F_PL7


title: b110: deplete_AQUIL_T0 E


title: Rpph1_control_NAI

city: Gainesville

first: Maurice

middle: S

last: Swanson

state: FL

address: 2033 Mowry Road,, Cancer & Genetics Research Complex, Swanson Lab, Room 220G

institute: University of Florida

description:
extracted fastq
rpph1.map

data_processing:
Library strategy: SHAPE-MaP
Fastq files were obtained from Illumina
reads were trimmed and filtered to a uniform length of 70 basepairs using bbduk
Fastq files were used for RNA structure mapping with Shapemapper2(v2.1.3)
The DMSO sequences are for the ShapeMapper program as the untreated control.
Genome_build: mm10
Supplementary_files_format_and_content: .map files from Shapemapper2 describing reactivity at each position of the target sequence. column 1 is 1-based nucleotide position, column 2 is normalized reactivity, column 3 is stderr and column 4 is nucleotide sequence.

source_name: fibroblast

growth_protocol: Mbnl1-/-/Mbnl2-/- MEFs were routinely cultured in DMEM with 10% FBS, 1x Pen/Strep

extract_protocol:
Total RNA was extracted using the DirectZol RNA Miniprep Plus kit from Zymo, including an on-column DNAse digestion step.
SHAPE-Map libraries were constructed as discussed in the paper.

treatment_protocol: To induce MBNL1 expression cells were induced with 1 ug/mL of doxycycline for 18-20 hours.  Cells well harvested with TyrpLE and rinsed with PBS.  Aliquots of 15 x 10^6 cells were treated with 100 mM NAI or DMSO only as a control for 12 minutes before total RNA extraction.

tag: cell type

value: MEFs

tag: genotype

tag: treatment

value: NAI

tag: target sequence


title: Rpph1_control_DMSO

description:
extracted fastq
rpph1.map

value: DMSO


title: b109: deplete_AQUIL_T0 D


title: Rn7SK_control_NAI

description:
extracted fastq
Rn7SK.map


title: MBNLamplicon_Dox_NAI_Set3

description:
amplicon
MBNL1Dox_3.map


title: MBNLamplicon_Dox_NAI_Set2

description:
amplicon
MBNL1Dox_2.map


title: b107: deplete_L_T4 E


title: b106: deplete_L_T2 C


title: MBNLamplicon_DMSO_Set4

description: amplicon


title: MBNLamplicon_No_dox_NAI_Set1

description:
amplicon
MBNL1No_dox_1.map


title: b105: deplete_AQUIL_T4 A


title: MBNLamplicon_No_dox_NAI_Set2

description: amplicon


title: TZPL03_Placenta of control 6-somite embryo  (1xJ cross) #3

description:
CONTROL.SIX.1J.3
CONTROL_WT_1J_6SOM_M_PL4

tag: sample type

tag: embryo genotype

tag: genotype of maternal germ line

tag: genotype of father


title: MBNLamplicon_DMSO_Set1

description: amplicon


title: MBNLamplicon_DMSO_Set3

description: amplicon


title: MBNLamplicon_DMSO_Set2

description: amplicon


title: b108: deplete_AQUIL_T0 A


title: MBNLamplicon_Dox_NAI_Set1

description:
amplicon
MBNL1Dox_1.map


title: b111: deplete_NOADD_Tm1 C

value: NOADD


title: TZPL01_Placenta of control 6-somite embryo (1xJ cross) #1

description:
CONTROL.SIX.1J.1
CONTROL_WT_1J_6SOM_F_PL1

tag: somite number

tag: cross

tag: cross abbreviation

tag: Sex

value: female


title: Rn7SK_control_DMSO

description:
extracted fastq
Rn7SK.map


title: b102: deplete_L_T2 D


title: Maternal-zygotic Ehmt2 mutant 4-somiteembryo (1XJ cross) #1

description:
MATZYG.FOUR.1J.1
MATZYG_HOMO_1J_4SOM_F_EM58

source_name: 4-somite embryo

extract_protocol:
Total RNA was isolated from embryos by acid-phenol extraction using RNA-Bee (Tel-TEST) followed by isopropanol precipitation. Genomic DNA was removed with the DNA-free Kit (Ambion).
Total RNA ibraries were prepared by the Van Andel Genomics Core from 500 ng of total RNA using the KAPA RNA HyperPrep Kit with RiboseErase (v1.16) (Kapa Biosystems, Wilmington, MA USA) following kit parameters. In short, RNA was sheared for 6 min at 86 oC to produce 300-400 bp fragments and it was converted to cDNA. After repairing the ends the cDNA fragments were ligated to Bioo Scientific NEXTflex Adapters (Bioo Scientific, Austin, TX, USA) and amplified using 11 cycles of PCR. Quality and quantity of the finished libraries were assessed using a combination of Agilent DNA High Sensitivity chip (Agilent Technologies, Inc.), QuantiFluor® dsDNA System (Promega Corp., Madison, WI, USA), and Kapa Illumina Library Quantification qPCR assays (Kapa Biosystems).  Individually indexed libraries were pooled, and paired end 75 bp sequencing was performed on an Illumina NextSeq 500 sequencer using a 150 bp HO sequencing kit (v2) (Illumina Inc., San Diego, CA, USA), with all libraries run across 5 flowcells to return a minimum read depth of 40M read pairs per library. Base calling was done by Illumina NextSeq Control Software (NCS) v2.0 and output of NCS was demultiplexed and converted to FastQ format with Illumina Bcl2fastq v1.9.0.
Allele-specific, stranded, paired-end total RNA seq

treatment_protocol: An Ehmt2 conditional knockout mouse line (Ehmt2fl/fl) was generated in our laboratory by gene targeting in 129S1/SvImJ ES cells (Zeng, Han et al. PNAS 2019). Ehmt2fl/fl and Ehmt2+/−,129S mice were maintained on 129S1 background. The floxed SET domain was removed to generate the Ehmt2−(129S1) allele by crossing the Ehmt2fl/+ male with 129S1/Sv-Hprttm1(CAGcre)Mann/J transgenic mouse female (Tang, Silva et al. 2002). Embryos were dissected between 8.5 dpc and 9.5 dpc, and were genotyped for Ehmt2 mutation status and for sex by PCR using their allantois as described (Zeng, Han et al. 2019). Ehmt2−/− homozygous and Ehmt2+/+ wild type embryos were obtained by natural mating of Ehmt2+/− females and males. To distinguish the paternal and maternal alleles, we used F1 hybrid samples collected from reciprocally crossedparents from the inbred mouse strains 129S1 (Mus musculus musculus) and JF1/Ms (Mus musculus molossinus) (JF1). We backcrossed the Ehmt2fl(129S1) allele to the JF1 mouse strain for more than ten generations to obtaine the Ehmt2−(JF1.N10) allele, where the genome (except the Ehmt2 locus and its close neighbors, in the approximate interval of chr 17:34647146-35241746) has been replaced by JF1 chromosomes during meiotic recombination events. Crossing a female carrying this allele with the original Ehmt2−(129S1) male yielded the Ehmt2−(JF1.N10)/−(129S1) zygotic mutant and Ehmt2+(JF1)/+(129S1) wild type embryos. The reciprocal cross resulted in Ehmt2−(129S1)/−(JF1.N10) mutant and Ehmt2+(129S1)/+(JF1) wild type embryos. Control Ehmt2+(129S1)/+(JF1) embryos were obtained from wild typel JF1 and 129S1 mice. Ehmt2fl/fl; Zp3-CreTg/0 females were crossed with wild type JF1 males to generate Ehmt2mat−/+JF1 embryos. Their control came from crossing Ehmt2fl/fl; Zp3-CreTg- females with wild type JF1 males. Ehmt2fl/fl; Zp3-CreTg/0 females were crossed with Ehmt2+/−(JF1.N10) males to generate Ehmt2mat−/zyg-JF1 embryos. Ehmt2+/- (JF1.N10) dams and wild-type 129S1 sires yielded maternal haploinsufficient wild type embryos. Ehmt2+/-(129S1) sire and JF1 dam yielded paternal haploinsufficient wild type embryos.

value: MATERNAL-ZYGOTIC MUTANT

value: homozygous mutant

value: HOMO (fl/fl;Cre+)

value: FOUR

tag: age (dpc) adjusted to cross

tag: day of collection (gestational day)


title: Maternal-zygotic Ehmt2 mutant 6-somite embryo (1XJ cross) #2

description:
MATZYG.SIX.1J.2
MATZYG_HOMO_1J_6SOM_F_EM64

source_name: 6-somite embryo

value: heterozygous mutant

value: 129SvImJ x JF1/MsJ

tag: time of collection


title: Wild type 6-somite embryo (Jx1 cross) #4

description:
WT.SIX.J1.4
ZYG_WT_J1_6SOM_M_EM72

value: WILD TYPE

value: wild type


title: b101: deplete_L_T2 B

tag: iron bioavailability

tag: media


title: Wild type 6-somite embryo (Jx1 cross) #3

description:
WT.SIX.J1.3
ZYG_WT_J1_6SOM_M_EM61

value: SIX

value: JF1/MsJ x 129SvImJ


title: Wild type 12-somite embryo (Jx1 cross) #4

description:
WT.TWELVE.J1.4
ZYG_WT_J1_12SOM_M_EM72Dec

source_name: 12-somite embryo

value: TWELVE

value: male


title: Wild type 6-somite embryo (Jx1 cross) #1

description:
WT.SIX.J1.1
ZYG_WT_J1_6SOM_F_EM71


title: Wild type 6-somite embryo (Jx1 cross) #2

description:
WT.SIX.J1.2
ZYG_WT_J1_6SOM_F_EM89


title: b100: deplete_NOADD_Tm1 B

value: deplete

tag: exposure time


title: Wild type 12-somite embryo (Jx1 cross) #2

description:
WT.TWELVE.J1.2
ZYG_WT_J1_12SOM_F_EM73


title: b104: deplete_AQUIL_T2 C

value: AQUIL


title: b099: deplete_L_T1 A

value: L


title: b103: deplete_AQUIL_T1 C


title: b098: deplete_L_T1 B


title: Wild type 12-somite embryo (Jx1 cross) #1

description:
WT.TWELVE.J1.1
ZYG_WT_J1_12SOM_F_EM38


title: Wild type 6-somite embryo (1xJ cross) #4

description:
WT.SIX.1J.4
ZYG_WT_1J_6SOM_M_EM39


title: b097: deplete_L_T1 E


title: b095: deplete_AQUIL_T2 D


title: b096: deplete_AQUIL_T3 C


title: Wild type 6-somite embryo (1xJ cross) #3

description:
WT.SIX.1J.3
ZYG_WT_1J_6SOM_M_EM22


title: b094: deplete_AQUIL_T3 D


title: Wild type 6-somite embryo (1xJ cross) #2

description:
WT.SIX.1J.2
ZYG_WT_1J_6SOM_F_EM475


title: Wild type 6-somite embryo (1xJ cross) #1

description:
WT.SIX.1J.1
ZYG_WT_1J_6SOM_F_EM44


title: b093: deplete_L_T3 B


title: Homozygous Ehmt2 mutant 6-somite embryo (Jx1 cross) #3

description:
HOMO.SIX.J1.3
ZYG_HOMO_J1_6SOM_M_EM35

value: ZYGOTIC HOMOZYGOUS MUTANT


title: b092: deplete_AQUIL_T2 A


title: Homozygous Ehmt2 mutant 12-somite embryo (Jx1 cross) #4

description:
HOMO.TWELVE.J1.4
ZYG_HOMO_J1_12SOM_M_EM41


title: Wild type 12-somite embryo (Jx1 cross) #3

description:
WT.TWELVE.J1.3
ZYG_WT_J1_12SOM_M_EM70Dec


title: Homozygous Ehmt2 mutant 12-somite embryo (Jx1 cross) #1

description:
HOMO.TWELVE.J1.1
ZYG_HOMO_J1_12SOM_F_EM47

value: female


title: Homozygous Ehmt2 mutant 6-somite embryo (1XJ cross) #3

description:
HOMO.SIX.1J.3
ZYG_HOMO_1J_6SOM_M_EM59


title: Homozygous Ehmt2 mutant 6-somite embryo (Jx1 cross) #1

description:
HOMO.SIX.J1.1
ZYG_HOMO_J1_6SOM_F_EM68


title: Homozygous Ehmt2 mutant 6-somite embryo (Jx1 cross) #2

description:
HOMO.SIX.J1.2
ZYG_HOMO_J1_6SOM_F_EM68Dec


title: Homozygous Ehmt2 mutant 12-somite embryo (Jx1 cross) #2

description:
HOMO.TWELVE.J1.2
ZYG_HOMO_J1_12SOM_F_EM48


title: Homozygous Ehmt2 mutant 12-somite embryo (Jx1 cross) #3

description:
HOMO.TWELVE.J1.3
ZYG_HOMO_J1_12SOM_M_EM40


title: Homozygous Ehmt2 mutant 6-somite embryo (1XJ cross) #2

description:
HOMO.SIX.1J.2
ZYG_HOMO_1J_6SOM_F_EM15


title: Homozygous Ehmt2 mutant 6-somite embryo (1XJ cross) #4

description:
HOMO.SIX.1J.4
ZYG_HOMO_1J_6SOM_M_EM82


title: b091: deplete_NOADD_Tend A

value: NOADD

value: Tend


title: Homozygous Ehmt2 mutant 6-somite embryo (1XJ cross) #1

description:
HOMO.SIX.1J.1
ZYG_HOMO_1J_6SOM_F_EM13

value: homozygous mutant


title: Maternal Ehmt2 haploinsufficient 6-somite embryo(Jx1 cross) #4

description:
MATHAPLO_WT.SIX.J1.4
MATHAPLO_WT_J1_6SOM_M_EM21

value: MATERNAL HAPLOINSUFFICIENT WILD TYPE


title: b090: deplete_L_T2 A


title: b089: deplete_AQUIL_T4 E


title: Maternal Ehmt2 haploinsufficient 6-somite embryo(Jx1 cross) #1

description:
MATHAPLO_WT.SIX.J1.1
MATHAPLO_WT_J1_6SOM_F_EM18

value: heterozygous mutant


title: Maternal Ehmt2 haploinsufficient 6-somite embryo(Jx1 cross) #2

description:
MATHAPLO_WT.SIX.J1.2
MATHAPLO_WT_J1_6SOM_F_EM24


title: b088: deplete_L_T3 A


title: Maternal Ehmt2 haploinsufficient 6-somite embryo(Jx1 cross) #3

description:
MATHAPLO_WT.SIX.J1.3
MATHAPLO_WT_J1_6SOM_M_EM20


title: Paternal Ehmt2 haploinsufficient 6-somite embryo (Jx1 cross) #4

description:
PATHAPLO_WT.SIX.J1.4
PATHAPLO_WT_J1_6SOM_M_EM30

value: PATERNAL HAPLOINSUFFICIENT WILD TYPE

value: WT


title: b087: deplete_L_T2 E


title: Paternal Ehmt2 haploinsufficient 6-somite embryo (Jx1 cross) #1

description:
PATHAPLO_WT.SIX.J1.1
PATHAPLO_WT_J1_6SOM_F_EM26

value: wild type


title: Paternal Ehmt2 haploinsufficient 6-somite embryo (Jx1 cross) #3

description:
PATHAPLO_WT.SIX.J1.3
PATHAPLO_WT_J1_6SOM_M_EM29

value: SIX


title: Maternal control 6-somite embryo (1xJ cross) #4

description:
MATWT.SIX.1J.4
MAT_WT_1J_6SOM_M_EM7

value: MATERNAL CONTROL WILD TYPE

value: WT (fl/fl;Cre-)

value: 129SvImJ x JF1/MsJ


title: Paternal Ehmt2 haploinsufficient 6-somite embryo (Jx1 cross) #2

description:
PATHAPLO_WT.SIX.J1.2
PATHAPLO_WT_J1_6SOM_F_EM34

value: JF1/MsJ x 129SvImJ


title: Maternal control 6-somite embryo (1xJ cross) #2

description:
MATWT.SIX.1J.2
MAT_WT_1J_6SOM_F_EM8


title: b085: deplete_L_T3 D

value: deplete


title: Maternal control 6-somite embryo (1xJ cross) #3

description:
MATWT.SIX.1J.3
MAT_WT_1J_6SOM_M_EM6

value: male


title: Maternal control 6-somite embryo (1xJ cross) #1

description:
MATWT.SIX.1J.1
MAT_WT_1J_6SOM_F_EM5


title: b086: deplete_L_T4 C

value: L


title: Maternal Ehmt2 mutant 6-somite embryo (1xJ cross) #4

description:
MATMUT.SIX.1J.4
MAT_MUT_1J_6SOM_M_EM50

value: MATERNAL MUTANT

value: homozygous mutant (fl/fl;Cre+)


title: Homozygous Ehmt2 mutant 6-somite embryo (Jx1 cross) #4

description:
HOMO.SIX.J1.4
ZYG_HOMO_J1_6SOM_M_EM85

value: ZYGOTIC HOMOZYGOUS MUTANT


title: Maternal Ehmt2 mutant 6-somite embryo (1xJ cross) #2

description:
MATMUT.SIX.1J.2
MAT_MUT_1J_6SOM_F_EM48

value: female


title: Maternal Ehmt2 mutant 6-somite embryo (1xJ cross) #3

description:
MATMUT.SIX.1J.3
MAT_MUT_1J_6SOM_M_EM44


title: b081: deplete_L_T0 C


title: b084: deplete_AQUIL_T4 C

value: AQUIL


title: b083: deplete_AQUIL_T3 E


title: b080: deplete_AQUIL_T1 B


title: Maternal Ehmt2 mutant 6-somite embryo (1xJ cross) #1

description:
MATMUT.SIX.1J.1
MAT_MUT_1J_6SOM_F_EM10

value: heterozygous mutant


title: Control 6-somite embryo (Jx1 cross) #2

description:
CONTROL.SIX.J1.2
CONTROL_WT_J1_6SOM_F_EM24

value: CONTROL


title: Control 6-somite embryo (Jx1 cross) #1

description:
CONTROL.SIX.J1.1
CONTROL_WT_J1_6SOM_F_EM19


title: b079: deplete_AQUIL_T3 B


title: Control 6-somite embryo (1xJ cross) #3

description:
CONTROL.SIX.1J.3
CONTROL_WT_1J_6SOM_M_EM4


title: b078: deplete_NOADD_Tm1 D

value: NOADD


title: Control 6-somite embryo (1xJ cross) #1

description:
CONTROL.SIX.1J.1
CONTROL_WT_1J_6SOM_F_EM2

value: wild type


title: Control 6-somite embryo (1xJ cross) #2

description:
CONTROL.SIX.1J.2
CONTROL_WT_1J_6SOM_F_EM9

value: SIX


title: Control 6-somite embryo (1xJ cross) #4

description:
CONTROL.SIX.1J.4
CONTROL_WT_1J_6SOM_M_EM6


title: A53T_Brain_4

city: Wuerzburg

first: Rhonda

last: McFleder

address: Josef-schneider-strasse 11

institute: Neurologische Klinik und Poliklinik des Universitaetsklinikum Wuerzburg

data_processing:
Quality and adaptor sequence trimmed via Cutadapt v. 2.5 using a cutoff Phred score of 20 in NextSeq mode and reads without any remaining bases were discarded; Parameters:  --nextseq-trim=20 -m 1 -a NNNAGATCGGAAGAGCACACGTCTGAACTCCAGTCAC -O 6
Trimmed reads were mapped to the mm10 mouse genome and human SNCA A53T using STAR v2.7.2b.
Read counts generated using featureCounts v1.6.4. Multi-mapping and multi-overlapping reads were counted strand-specific and reversely stranded with a fractional count for each alignment and overlapping feature; Parameters: -s 2 -t exon -M -O --fraction
DESeq2 version 1.24.0 was utilized to normalize read counts, to generate regularized log2-transformed read counts, and to perform differential gene expression analysis with fold-change shrinkage by setting the parameter to "betaPrior=True"
Genome_build: mm10 mouse genome (GRCm38.p6 primary assembly and mitochondrion)
Supplementary_files_format_and_content: raw_count_table
Supplementary_files_format_and_content: rlog-transformed_count_table

growth_protocol: Only male C57BL/6  mice were used maintained in a specific pathogen-free environment under standard conditions of the animal facility of the University Hospital of Würzburg

extract_protocol:
Substantia Nigra and striatum were microdissected, incubated for 30 min at 37°C and 650 rpm in 1mL HBSS with 5 mg Collagenase NB4 and 0.1 mg DNaseI followed by the generation of cell suspensions in HBSS with a 70 µm cell strainer. Cells were centrifuged at 240 g and 18°C for 5 min. The pellet was resuspended in 30% Percoll/PBS and immune cells were isolated after centrifugation at 650G and 18°C for 30 min with a 70% Percoll/PBS gradient. RNA isolation was performed with the RNeasy micro kit (Qiagen cat # 74004) according to manufacturer's instructions.
Libraries were prepared from 0.5 ng of total RNA using the SMARTer Stranded Total RNA-Seq Kit v2 (Takara Bio cat#634411) with 4 min RNA fragmentation at 94°C, 5 cycles of PCR for cDNA library pre-amplification and 15 cycles of PCR amplification after rDNA depletion.

treatment_protocol: 10-12 week old mice were injected with an AAV1/2 virus with or without A53T haSyn 16 weeks prior to RNA isolation; 26 - 28 week old, male mice at the day of RNA isolation

tag: strain background

tag: aav virus

value: A53T haSyn

tag: cell type

value: Immune cells in Substantia Nigra and Striatum


title: A53T_Brain_3


title: Control 6-somite embryo (Jx1 cross) #4

description:
CONTROL.SIX.J1.4
CONTROL_WT_J1_6SOM_M_EM23

tag: sample type


title: b077: deplete_L_T3 C


title: b082: deplete_L_T4 D


title: b076: deplete_AQUIL_T0 C


title: Control 6-somite embryo (Jx1 cross) #3

description:
CONTROL.SIX.J1.3
CONTROL_WT_J1_6SOM_M_EM20

tag: embryo genotype


title: A53T_Brain_2


title: EV_Brain_3

value: Empty Vector


title: A53T_Brain_1


title: EV_Brain_4


title: b075: deplete_L_T0 E


title: b073: deplete_AQUIL_T1 A


title: b074: deplete_NOADD_Tm1 E


title: EV_Brain_1


title: EV_Brain_2


title: A53T_Brain_5


title: b071: deplete_AQUIL_T1 D


title: HA-a1-4: E5_Chick_Ceca 2

city: Boston

first: Rhian

last: Stavely

state: MA

address: 185 Cambridge Street, CPZN, 6100

department: Pediatric Surgery

institute: Massachusetts General Hospital

data_processing:
RNA-seq libraries were sequenced on an Illumina 2500 instrument resulting in 20-30 million reads per sample
Base calling was performed using HiSeq Control Software and sequencing data were generated using the Illumina bcl2fastq pipeline.
Sequence reads were aligned to the Gallus gallus reference genome (Galgal4) using the splice-aware alignment program STAR
Read summarization of the counts per gene was determined using the program HTSeq
Differential expression of transcripts between the ceca and interceca were determined using the R package edgeR.
Genome_build: Galgal4
Supplementary_files_format_and_content: Processed data contains count output in RPKM

organism: Gallus gallus

source_name: Embryonic gut

extract_protocol:
The gut was harvested from E5 chick embryos, the ceca and interceca regions were carefully dissected and RNA was extracted from the tissues using Trizol reagent as of manufacturers instructions.
Libraries were constructed using the NEBNext Ultra Directional RNA Library Prep Kit for Illumina as of manufacturers protocol.

tag: developmental stage

tag: tissue

value: Ceca


title: HA-a1-5: E5_Chick_Interceca 3

value: Interceca


title: HA-a1-6: E5_Chick_Ceca 3


title: b072: deplete_AQUIL_T1 E

value: deplete


title: EV_Brain_5

value: male


title: 3P2-9-44_S97

city: Rensselaer

first: Nathan

middle: C

last: Boles

address: One Discovery Dr

institute: Neural Stem Cell Institute

data_processing:
After de-multiplexing fastq files were mapped with STAR aligner (version 2.7.0a)
A raw counts matrix was generated in R with the GenomicAlignments package.
Genome_build: mm10
Supplementary_files_format_and_content: Comma-seperated file of raw counts with samples by column

source_name: meninges

extract_protocol:
Meninges were dissected from the anterior and posterior of the cortex of  C57BL/6 mice and a single cell suspension was generated. Cells were dispensed using the ICELL8 single cell system as single cells followed by reverse transcription and barcoding according to the manufacturer's protocol
RNA libraries were prepared for sequencing the 150 cycle Nextera DNA library preperation kit from Illumina.

value: Posterior meninges

tag: age

value: 3 months


title: 3P2-52-42_S103


title: 3P2-53-39_S100


title: 3P2-53-46_S99


title: 3P2-53-23_S101


title: 3P2-52-39_S104


title: b070: deplete_NOADD_Tend B

value: Tend


title: 3P2-9-18_S98


title: 3P2-52-38_S105


title: b069: deplete_NOADD_Tm1 A


title: HA-a1-3: E5_Chick_Interceca 2


title: HA-a1-2: E5_Chick_Ceca 1


title: b068: deplete_AQUIL_T0 B


title: 3P2-52-19_S108


title: HA-a1-1: E5_Chick_Interceca 1


title: 3P2-52-21_S107


title: 3P2-52-37_S106


title: b067: deplete_AQUIL_T3 A


title: 3P2-51-70_S110


title: 3P2-51-37_S111


title: Methylome ibm1 met1 epiRIL12

city: Athens

first: Robert

middle: J

last: Schmitz

state: GA

address: B416 Davison Life Sciences

department: Genetics

institute: University of Georgia

data_processing:
All WGBS data used in this study were processed by Methylpy v1.3 as described in (Schultz, He et al. 2015).
Quality filtering and adapter trimming were performed using cutadapt v1.9.dev1 (Martin 2011).
Qualified reads were aligned to the A. thaliana TAIR10 reference genome (Berardini, Reiser et al. 2015) (downloaded from https://phytozome.jgi.doe.gov) using bowtie 2.2.4 (Langmead and Salzberg 2012).Only uniquely aligned and nonclonal reads were retained.
Chloroplast DNA (which is fully unmethylated) was used as a control to calculate the sodium bisulfite reaction nonconversion rate of unmodified cytosines. A binomial test was used to determine the methylation status of cytosines with a minimum coverage of three reads.
Genome_build: A. thaliana TAIR10 reference genome (Berardini, Reiser et al. 2015) (downloaded from https://phytozome.jgi.doe.gov)
Supplementary_files_format_and_content: allc file which record the mapped reads number for each C site.

organism: Arabidopsis thaliana

source_name: ibm1 met1 epiRIL12

growth_protocol: Seeds were planted and grown in 16-hour  day lengths and samples were harvested from young  above ground tissue. Tissue was flash frozen in liquid  nitrogen and DNA was isolated using a Qiagen  Plant DNeasy kit (Qiagen, Valencia, CA, USA) according  to the manufacturer’s instructions.

extract_protocol:
MethylC-seq: prepared as previously described in (Urich et al. 2015).
MethylC–seq

value: Young leaf tissue


title: b066: deplete_L_T0 A

value: L


title: 3P2-52-61_S102


title: 3P2-50-20_S112


title: 3P2-49-40_S113


title: 3P2-49-20_S114


title: b065: replete_L_T3 D

value: replete


title: ChIP-seq_Mcm_IP_D_strain-40

first: Fiona

last: Jenkinson

address: Tennis court road

department: Biochemistry

institute: University of Cambridge

data_processing:
Sequence reads were mapped using Bowtie2 (version 2.2.6).
Repli-Seq - Unique reads mapping to a single genomic location were summed in 1 kb bins, using Samtools (version 1.7.2) and Bedtools (version 2.26.0).
ChIP-Seq - IP samples and correspoding input BAM files were compared using bamCompare (version 3.0.2). To  compare the BAM files, the genome is partitioned into bins of equal size, then  the number of reads found in each bin is counted per file, and finally the log2 ratio is reported. The IP and input are normalised for sequencing depth.
Genome_build: sacCer3
Supplementary_files_format_and_content: Repli-Seq - csv files containing number of unique reads in 1 kb bins normalised to G1 controls.
Supplementary_files_format_and_content: ChIP-Seq - bigWig files containing containing the bin location and the log2 ratio of the IP over input (normalised to sequencing depth)

organism: Saccharomyces cerevisiae

source_name: S-phase yeast cells - 40 min after G1 release

growth_protocol: Mid log phase yeast culture grown in standard media (YPAD ) in synchronous progression from G1 phase

extract_protocol:
For Repli-seq, DNA was purified by phenol chloroform extraction. For CHIP DNA was extracted using beadbeating, IP, SDS elution and purified with CHIP clean concentrator kit (Zymo).
Library preparation was carried out according to Illumina TruSeq Nano instructions for Repli-seq. For CHIP-seq: Sequencing libraries were prepared according to instructions of Illumina Truseq-Nano except: size selection was adjusted for 200bp fragments; adaptors were used at 1:100 dilution and 14-16 cycles of amplification were performed.

treatment_protocol: Yeast culture at 1x10^7 cells/ml were arrested and maintained in G1 with 5ug/ml alpha factor, upon arrest of >90% of cells 1mM IAA was added with a 30 minute temperature shift to 37 degrees C to induce degradation of cdc55-aid and pph3-aid, cells were then washed and released synchronously into fresh YPAD medium containing 1mM IAA. 5ml samples were taken and mixed with Sodium Azide/EDTA for Repli-seq. 200ml samples were taken for CHIP-seq, fixing with 1% formaldehyde for 25mins (Mcm) or 1.5mM EGS for 10mins followed by 1% Formaldehyde for 10 mins (Sld2). Crosslinking was quenched in 125mM Glycine.

tag: strain

value: swe1delta pph3-AID cdc55-AID sld3-r-13myc

tag: genotype

value: MATa swe1delta::TRP1 cse4delta::NatMX leu2::CSE4-HA6::LEU2 CDC55-AID::KanMx PPH3-AID::HphMx his3::PGPD1-osTir1::HIS3 sld3-R(2A)-13myc::KanMx Mcm4-3HA::KanMX

tag: antibody

value: Mcm


title: ChIP-seq_Mcm_IP_D_strain-25

source_name: S-phase yeast cells - 25 min after G1 release


title: 3P2-49-19_S115


title: b064: deplete_AQUIL_T2 B

value: AQUIL


title: 3P2-48-66_S117


title: ChIP-seq_Mcm_IP_D_strain-G1

source_name: Synchronised G1 phase yeast cells


title: 3P2-49-12_S116


title: 3P2-48-65_S118


title: b062: replete_AQUIL_T3 B


title: 3P2-52-13_S109


title: b063: deplete_L_T1 D


title: ChIP-seq_Mcm_input_D_strain-25


title: 3P2-47-46_S120


title: 3P2-47-18_S121


title: 3P2-46-62_S123


title: ChIP-seq_Mcm_input_D_strain-G1


title: b060: replete_L_T1 D


title: 3P2-48-18_S119


title: b061: replete_L_T2 C


title: 3P2-46-71_S122


title: ChIP-seq_Mcm_input_D_strain-40


title: 3P2-46-15_S124


title: 3P2-45-46_S125


title: ChIP-seq_Mcm_IP_A_strain-40

value: swe1delta cdc55-AID sld3-13myc

value: MATa swe1delta::TRP1 cse4delta::NatMX leu2::CSE4-HA6::LEU2 CDC55-AID::KanMx his3::PGPD1-osTir1::HIS3 SLD3-13myc::KanMx Mcm4-3HA::KanMX


title: b059: replete_AQUIL_T4 A


title: 3P2-45-20_S126


title: ChIP-seq_Mcm_IP_A_strain-25


title: 3P2-17-71_S127


title: 3P2-16-13_S128


title: ChIP-seq_Mcm_IP_A_strain-G1


title: 3P2-15-65_S129


title: 3P2-15-60_S130


title: b057: replete_L_T2 D


title: ChIP-seq_Mcm_input_A_strain-40


title: 3P2-15-41_S131


title: 3P2-15-20_S132


title: b056: replete_AQUIL_T1 E


title: ChIP-seq_Mcm_input_A_strain-25


title: 3P2-14-64_S133


title: ChIP-seq_Mcm_input_A_strain-G1


title: b055: replete_L_T3 C


title: 3P2-14-39_S134


title: 3P2-14-14_S135


title: b054: deplete_AQUIL_T4 D


title: RepTime_D_strain-80

data_processing:
Library strategy: Repli-Seq
Sequence reads were mapped using Bowtie2 (version 2.2.6).
Repli-Seq - Unique reads mapping to a single genomic location were summed in 1 kb bins, using Samtools (version 1.7.2) and Bedtools (version 2.26.0).
ChIP-Seq - IP samples and correspoding input BAM files were compared using bamCompare (version 3.0.2). To  compare the BAM files, the genome is partitioned into bins of equal size, then  the number of reads found in each bin is counted per file, and finally the log2 ratio is reported. The IP and input are normalised for sequencing depth.
Genome_build: sacCer3
Supplementary_files_format_and_content: Repli-Seq - csv files containing number of unique reads in 1 kb bins normalised to G1 controls.
Supplementary_files_format_and_content: ChIP-Seq - bigWig files containing containing the bin location and the log2 ratio of the IP over input (normalised to sequencing depth)

source_name: S-phase yeast cells - 80 min after G1 release

value: MATa swe1delta::TRP1 cse4delta::NatMX leu2::CSE4-HA6::LEU2 CDC55-AID::KanMx PPH3-AID::HphMx his3::PGPD1-osTir1::HIS3 sld3-R(2A)-13myc::KanMx


title: 3P2-12-69_S137


title: RepTime_D_strain-70

source_name: S-phase yeast cells - 70 min after G1 release


title: 3P2-12-67_S138


title: b053: replete_AQUIL_T2 B


title: 3P2-12-17_S139


title: b058: replete_AQUIL_T3 C


title: 3P2-11-47_S140


title: 3P2-11-44_S141


title: b052: replete_AQUIL_T1 D


title: RepTime_D_strain-60

source_name: S-phase yeast cells - 60 min after G1 release


title: RepTime_D_strain-50

source_name: S-phase yeast cells - 50 min after G1 release


title: b051: replete_L_T2 A


title: 3P2-10-71_S143


title: b050: replete_L_T0 E


title: 3P2-10-63_S144


title: RepTime_D_strain-45

source_name: S-phase yeast cells - 45 min after G1 release


title: b049: replete_NOADD_Tm1 B

value: NOADD


title: 3P2-13-36_S136


title: 3P2-10-23_S147


title: b048: replete_L_T1 B


title: 3P2-10-14_S149


title: b047: replete_L_T4 A


title: 3P1-9-7_S150


title: 3P2-10-19_S148


title: RepTime_D_strain-35

source_name: S-phase yeast cells - 35 min after G1 release


title: 3P1-9-53_S151


title: 3P1-9-28_S153


title: 3P1-9-34_S152


title: RepTime_D_strain-25


title: 3P2-11-38_S142


title: b046: replete_L_T1 E

source_name: Bacterial culture


title: b045: replete_AQUIL_T1 A


title: 3P1-53-28_S154


title: RepTime_D_strain-20

source_name: S-phase yeast cells - 20min after G1 release


title: 3P1-53-0_S155


title: 3P2-10-62_S145


title: b044: replete_L_T1 A


title: 3P1-52-48_S157


title: 3P2-10-42_S146


title: RepTime_D_strain-40


title: b043: replete_AQUIL_T2 D


title: 3P1-52-30_S158


title: 3P1-51-8_S159


title: b042: replete_AQUIL_T4 B


title: RepTime_D_strain-30

source_name: S-phase yeast cells - 30 min after G1 release


title: RepTime_D_strain-G1


title: 3P1-51-28_S161


title: RepTime_C_strain-80

value: swe1delta pph3-AID cdc55-AID sld3-13myc

value: MATa swe1delta::TRP1 cse4delta::NatMX leu2::CSE4-HA6::LEU2 CDC55-AID::KanMx PPH3-AID::HphMx his3::PGPD1-osTir1::HIS3 SLD3-13myc::KanMx


title: b041: replete_NOADD_Tend C


title: 3P1-51-55_S160


title: RepTime_C_strain-70


title: 3P1-50-33_S164


title: RepTime_C_strain-60


title: 3P1-50-28_S166


title: b039: replete_NOADD_Tm1 E


title: 3P1-52-52_S156


title: RepTime_C_strain-50


title: RepTime_D_strain-15

source_name: S-phase yeast cells - 15 min after G1 release


title: b038: replete_L_T3 A


title: RepTime_C_strain-45


title: 3P1-49-34_S169


title: b037: replete_NOADD_Tm1 A


title: 3P1-49-29_S170


title: RepTime_C_strain-40


title: 3P1-49-24_S171


title: b036: replete_AQUIL_T0 C


title: 3P1-50-8_S162


title: 3P1-47-52_S172


title: 3P1-47-29_S173


title: 3P1-47-27_S174


title: 3P1-47-26_S175


title: RepTime_C_strain-30


title: 3P1-49-54_S167


title: 3P1-50-59_S163


title: b040: replete_AQUIL_T0 B


title: 3P1-46-55_S176


title: b035: replete_L_T4 E


title: b034: replete_AQUIL_T2 A


title: RepTime_C_strain-35


title: 3P1-46-30_S177


title: RepTime_C_strain-25


title: 3P1-50-32_S165


title: 3P1-45-30_S179


title: 3P1-45-54_S178


title: 3P1-49-49_S168


title: b032: replete_AQUIL_T0 E


title: RepTime_C_strain-20


title: 3P1-45-3_S180


title: b033: replete_AQUIL_T3 A


title: 3P1-17-54_S181


title: b031: replete_L_T0 D


title: RepTime_C_strain-15


title: 3P1-17-35_S183


title: 3P1-17-51_S182


title: RepTime_C_strain-G1


title: b030: replete_NOADD_Tend D


title: 3P1-16-57_S184


title: RepTime_B_strain-80

value: swe1delta cdc55-AID sld3-r-13myc

value: MATa swe1delta::TRP1 cse4delta::NatMX leu2::CSE4-HA6::LEU2 CDC55-AID::KanMx his3::PGPD1-osTir1::HIS3 sld3-R(2A)-13myc::KanMx


title: RepTime_B_strain-70


title: 3P1-16-3_S185

value: Posterior meninges

value: 3 months


title: 3P1-15-34_S186


title: b029: replete_AQUIL_T3 D


title: 3P1-15-25_S187


title: 3P1-14-59_S188


title: RepTime_B_strain-60


title: RepTime_B_strain-50


title: b027: replete_NOADD_Tm1 C


title: b028: replete_AQUIL_T3 E


title: b026: replete_AQUIL_T0 D


title: 3P1-13-29_S191


title: 3P1-13-52_S190


title: 3P1-12-54_S192


title: 3P1-12-33_S193


title: 3P1-11-8_S194


title: RepTime_B_strain-45


title: RepTime_B_strain-40


title: 3P1-11-56_S195


title: b024: replete_AQUIL_T2 E


title: b025: replete_L_T4 C


title: RepTime_B_strain-35


title: 3P1-11-34_S196


title: b023: replete_AQUIL_T4 D


title: 3P1-10-2_S197


title: 3A2-8-68_S198

value: Anterior meninges


title: RepTime_B_strain-30


title: 3A2-7-42_S199


title: 3A2-6-21_S200


title: 3A2-5-61_S201


title: b021: replete_AQUIL_T0 A


title: RepTime_B_strain-25


title: 3P1-13-56_S189


title: 3A2-43-16_S203


title: 3A2-41-36_S204


title: 3A2-44-42_S202


title: RepTime_B_strain-20


title: 3A2-39-65_S207


title: 3A2-39-43_S208


title: 3A2-37-17_S209


title: b018: replete_L_T1 C

value: L


title: 3A2-4-46_S206


title: RepTime_B_strain-15


title: RepTime_B_strain-G1


title: RepTime_A_strain-80

value: MATa swe1delta::TRP1 cse4delta::NatMX leu2::CSE4-HA6::LEU2 CDC55-AID::KanMx his3::PGPD1-osTir1::HIS3 SLD3-13myc::KanMx


title: b017: replete_L_T2 E

value: replete


title: 3A2-36-62_S210


title: 3A2-40-70_S205


title: b016: deplete_NOADD_Tend C

value: deplete

value: Tend


title: RepTime_A_strain-70


title: RepTime_A_strain-60


title: b022: replete_NOADD_Tend E


title: b015: deplete_NOADD_Tend D


title: 3A2-2-21_S212


title: 3A1-7-7_S214


title: 3A1-7-35_S215


title: b020: replete_L_T2 B


title: b014: replete_NOADD_Tm1 D


title: 3A1-6-57_S216


title: RepTime_A_strain-45


title: b019: replete_L_T0 C


title: 3A1-6-29_S217


title: b013: replete_AQUIL_T4 E

value: AQUIL


title: RepTime_A_strain-40

value: swe1delta cdc55-AID sld3-13myc


title: 3A1-6-1_S218


title: 3A1-5-52_S219


title: 3A2-36-44_S211


title: RepTime_A_strain-35


title: 3A1-5-11_S220


title: 3A1-43-11_S221


title: 3A1-41-5_S222


title: 3A1-8-35_S213


title: b012: replete_L_T0 A


title: RepTime_A_strain-30


title: b011: replete_L_T0 B


title: 3A1-41-11_S223


title: b010: replete_AQUIL_T1 C


title: RepTime_A_strain-25


title: RepTime_A_strain-50


title: 3A1-40-29_S225


title: 3A1-40-6_S224


title: RepTime_A_strain-20


title: b009: replete_AQUIL_T2 C


title: 3A1-39-0_S226


title: b008: replete_AQUIL_T4 C


title: 3A1-37-29_S229


title: RepTime_A_strain-15


title: 3A1-38-33_S227


title: 3A1-37-58_S228


title: b007: replete_L_T3 B


title: 3A1-37-26_S230


title: RepTime_A_strain-G1


title: 3A1-3-4_S231


title: b006: replete_L_T4 B


title: 3A1-0-55_S232


title: b005: replete_L_T3 E


title: 18P2-69-66_S233

value: 18 months


title: 18P2-68-40_S235


title: b004: replete_AQUIL_T1 B


title: 18P2-68-64_S234


title: 18P2-67-71_S236


title: 18P2-67-46_S237


title: b003: replete_NOADD_Tend A


title: 18P2-66-68_S238


title: b002: replete_NOADD_Tend B


title: 18P2-66-42_S240


title: 18P2-66-45_S239


title: 18P2-66-23_S241