title: scRNA+TCR-seq of tdTomato+ GFP+ CD4 T cells from the colonic lamina propria [TCR]

city: Princeton

first: Yuri

last: Pritykin

state: NJ

address: 245 Carl Icahn Lab, Lewis-Sigler Institute for Integrative Genomics

institute: Princeton University

description:
GFP_plus_Tom_plus
2548_GFP_plus_Tom_plus_rep1_2_3_TCR_IGO_11874_18_S17

data_processing:
FASTQ files from both scRNA and hashtag (HTO) sequencing were processed by “cellranger count” v6.0.1 using reference transcriptome refdata-gex-mm10-2020-A.
Analysis was then performed using scanpy v1.7.2. In each of the two conditions, HTO read counts for three hashtags were then used to assign each cell barcode to one of the three biological replicates. 19,449 cells with a total HTO read count > 300 and with at least 80% of the total HTO read count concentrated in one of the three hashtags were considered further as those with uniquely identifiable biological replicate assignment (filtering out 5,048 cells with uncertain or insufficient HTO data).
Furthermore, only cells with at least 100 captured genes and only genes captured in at least 10 cells were considered for subsequent analysis. In further preprocessing, data was normalized, non-protein-coding and ribosomal genes filtered out, cells with less than 300 captured genes and genes present in less than 10 cells removed, and likely contamination B and monocyte cells filtered out as a result of preliminary clustering analysis. This resulted in data with 5,100 cells in sample “GFP_minus_Tom_minus_rep1”, 3,067 in “GFP_minus_Tom_minus_rep2”, 1,815 in “GFP_minus_Tom_minus_rep3”, 1,725 in “GFP_plus_Tom_plus_rep1”, 4,560 in “GFP_plus_Tom_plus_rep2” and 2,391 cells in sample “GFP_plus_Tom_plus_rep3”.
This dataset was analyzed using PCA, kNN, UMAP, and PhenoGraph clustering.
For TCR reconstruction, FASTQ files from TCR sequencing were processed using “cellranger vdj” with reference refdata-cellranger-vdj-GRCm38-alts-ensembl-5.0.0 and otherwise default parameters.
Files filtered_contig_annotations.csv from cellranger vdj output for each sample were then used for TCR sequence and clonotype analysis using package scirpy v0.10.0 following the tutorial. Merging the TCR data with the scRNA-seq data and removing cells without at least one full pair of receptor sequences resulted in the dataset with 15,863 cells. Clonotypes were then defined using functions scirpy.pp.ir_dist() and scirpy. tl.define_clonotypes().
Genome_build: mm10
Supplementary_files_format_and_content: colon-cd4t-scrnaseq.csv is a scRNA-seq raw count matrix for the two samples, with three replicates each, combined in the CSV format. colon-cd4t-scrnaseq-cells.csv is a CSV file with metadata about cells. colon-cd4t-tcr.csv is a CSV file with metadata about cell TCR sequence analysis.

extract_protocol:
For isolation of CD4 T cells from the colon, tissues were enzymatically digested with DTT/EDTA buffer (PBS with 2mM L-glutamine, 10mM HEPES, 1x penicillin/streptomycin, 5% FCS, 1mM DTT and 2mM EDTA) for 15 minutes for 20 minutes at 37°C shaking at 250 RPM. Cells were filtered through a 100-µm nylon mesh to collect the intraepithelial fraction. Remaining colon tissue was further digested with Collagenase/DNAse buffer (RPMI-1640 with 2mM L-glutamine, 10mM HEPES, 1x penicillin/streptomycin, 5% FCS, 1 mg/ml collagenase A, and 1 µg/ml DNaseI) for 25 minutes at 37°C shaking at 250 RPM in the presence of five 0.25-inch ceramic spheres. Cells were filtered through a 100-µm nylon mesh to collect the lamina propria fraction. Cells were stained using fluorescent dye-conjugated antibodies and tdTomato+GFP+ and tdTomato-GFP- colonic lamina propria CD4 T cells were isolated using BD Aria II sorter.
Single-cell RNA-Seq of FACS-sorted cell populations was performed on a Chromium instrument (10X Genomics) following the user guide manual for 3′ v3 or 5′ v2 chemistry. In brief, FACS-sorted cells were washed once with PBS containing 0.04% bovine serum albumin (BSA) and resuspended in PBS containing 0.04% BSA to a final concentration of 700-1,200 cells per μl. The viability of cells was above 80%, as confirmed with 0.2% (w/v) Trypan Blue staining (Countess II). Cells were captured in droplets. Following reverse transcription and cell barcoding in droplets, emulsions were broken and cDNA was purified using Dynabeads MyOne SILANE (ThermoFisher: Cat # 37002D) followed by PCR amplification per manual instruction.
Approximately 5,000 cells were targeted for each sample, and triplicates from the same biological group were multiplexed together on one lane of 10X Chromium to reach 15,000 cells targeted, following cell hashing with TotalSeq-C reagents. ScRNA-seq and scTCR-seq libraries were prepared using the 10x Single Cell Immune Profiling Solution Kit, according to the manufacturer’s instructions. Briefly, amplified cDNA was used for both 5′ gene expression library construction and TCR enrichment. For gene expression library construction, amplified cDNA was fragmented and end-repaired, double-sided size-selected with SPRIselect beads, PCR-amplified with sample indexing primers (98 °C for 45 s; 14–16 cycles of 98 °C for 20 s, 54 °C for 30 s, 72 °C for 20 s; 72 °C for 1 min), and double-sided size-selected with SPRIselect beads. For TCR library construction, TCR transcripts were enriched from 2 μl of amplified cDNA by PCR (primer sets 1 and 2: 98 °C for 45 s; 10 cycles of 98 °C for 20 s, 67 °C for 30 s, 72 °C for 1 min; 72 °C for 1 min). Following TCR enrichment, enriched PCR product was fragmented and end-repaired, size-selected with SPRIselect beads, PCR-amplified with sample-indexing primers (98 °C for 45 s; 9 cycles of 98 °C for 20 s, 54 °C for 30 s, 72 °C for 20 s; 72 °C for 1 min), and size-selected with SPRIselect beads. Final libraries (GEX, TCR and HTO) were sequenced on Illumina NovaSeq 6000 platform (R1 – 26 cycles, i7 – 10 cycles, i5 – 10 cycles, R2 – 90 cycles).

value: CD4 T cells


title: iPSC neurons, wild type, day 14 of differentiation, rep4

status: Public on Jun 17 2022

type: SRA

city: Philadelphia

first: Alice

middle: S

last: Chen-Plotkin

state: PA

address: 150 Johnson Pavilion

department: Neurology

country: USA

institute: University of Pennsylvania

library_source: transcriptomic

data_processing:
QC assessed using FastQC and summarized with MultiQC.
Adapters removal and reads filtering by conducted by Trimmomatic Version 0.39 with following parameters: ILLUMINACLIP:TruSeq3-SE.fa:2:30:10 LEADING:5 TRAILING:5 SLIDINGWINDOW:5:20 MINLEN:50 AVGQUAL:30 HEADCROP:10
Alignment with Bowtie 2 with the following parameters: bowtie2 -q --phred33 --sensitive --dpad 0 --gbar 99999999 --mp 1,1 --np 1 --score-min L,0,-0.1 --nofw -p 32 -k 200 -x,
Read counts at the gene level were estimated by an expectation maximization algorithm built in RSEM.
Assembly: GRChg38
Supplementary files format and content: Matrix_of_raw_counts_per_gene_estimated_by_RSEM.txt: Matrix of raw read counts per gene estimated by RSEM. Each column is a sample and each row is a gene.

molecule: total RNA

organism: Homo sapiens

source_name: iPSC-N

growth_protocol: Human induced pluripotent stem cells (iPSC) with Neurog1 and Neurog2 in a bicistronic doxycycline-inducible expression cassette (provided by George Church lab) were used to generation of GPNMB wild-type (WT), heterozygote (Het), and knock-out (KO) iPSC and rapid induction of iPSC-derived neurons (iPSC-N). CRISPR knock out was achieved with sgRNA (Synthego) and recombinant Cas9 protein. iPSC cultures were maintained on Matrigel-coated 6cm tissue culture plates with mTeSR1 media and mechanically passaged every 4 days with StemMACS passaging solution XF. D0 iPSC-N were isolated 4 days after passaging, prior to neuronal induction. Neuronal induction was carried out by dissociating cells with Accutane, resuspending in N2 pre-differentiation media (KnockOut DMEM/F12 with 1x N-2 supplement, 1x MEM NEAA, 10ng/ml BDNF, 10ng/ml NT-3, 0.2ug/ml Laminin, and 2ug/ml doxycycline hyclate) with 10ug/ml Y-27632, and plating in matrigel-coated 6-well plates at 700k cells/well. Media was changed daily on days 1 and 2. On day 3, pre-differentiated iPSC-N were dissociated with accutase, resuspended in neuronal media (50% DMEM/F12, 50% Neurobasal-A media, 1x MEM NEAA, 0.5x N-2 supplement, 0.5x GlutaMAX supplement, 0.5x B27 supplement, 10ng/ml BDNF, 10ng/ml NT-3, 1ug/ml Laminin, and 2ug/ml doxycycline), and re-plated on PDL/matrigel coated glass coverslips at 200k cells/well in a 12-well plate format. Half-media changes were performed every 7 days and mature iPSC-N were harvested on D14 after induction.

extract_protocol:
RNA was extracted using QIAGEN RNeasy ® mini kit.
Library preparation was performed by the Penn Next Generation Sequencing (NGS) Core staff with the TruSeq Stranded mRNA Library Prep kit (Illumina cat #20020595) using 225ng total RNA and following manufacturer instructions.

tag: cell line

value: iPSC-N

tag: cell type

value: iPSC-derived neurons

tag: genotype

value: WT

tag: treatment

value: Doxycycline induced differentiation

tag: time point

value: Day 14

tag: replicate


title: Single-cell RNA-seq dataset of metamorphosed axolotl (Batch1), META1, sublibrary 18

city: Hangzhou

first: Fang

last: Ye

state: Zhejiang

address: 866 Yuhangtang Rd

country: China

institute: Zhejiang University

data_processing:
For single cell CH-RNA-seq. Sequenced reads were trimmed for adaptor sequence.Then reads in bam files were tagged with the cell and molecular (UMI) barcode sequences using SnapTools. The reads quality under 10 were removed. Cellbarcode:In read1, the bases 1-10,19-28 (NEO3,NEO4,META1,META2); the bases 1-10,42-51(NEO1,NEO2). UMI barcode:In read1, the bases 11-18(NEO3,NEO4,META1,META2); the bases 34-41(NEO1,NEO2).The raw fastq-format sequencing data from a DNBSEQ-T7 were first split into i7 indexed sub-libraries using splitBarcode (https://github.com/MGI-tech-bioinformatics/splitBarcode). For each sub-library, the revised Drop-seq core computational tool was used for data preprocessing  (http://mccarrolllab.org/wp-content/uploads/2016/03/Drop-seqAlignmentCookbookv1.2Jan2016.pdf). We used STAR (version 2.5.2a) with default parameters for mapping 3T3 cells and 293T cells. Reads from 3T3 cells and 293T cells were aligned to a merged hg19-mm10 reference genome (provided by Drop-seq group, GSE63269). To save mapping time , reads from axolotls were aligned to the axolotl genome (V3.0.0, https://www.axolotl-omics.org/assemblies) using another mapper bowtie. GTF annotation files were used to tag aligned reads. Cellular barcodes and unique molecular identifiers (UMIs) were directly extracted from Read1. A list of two rounds of cell barcode oligo sequences was used to correct the extracted cellular barcode from read1. Finally, the HTseq package was used to generate digital expression matrices of axolotl. For cell quality control of axolotl data, we excluded cells in which less than 200 transcripts were expressed. In the mixed-species experiment of 3T3 cells and 293T cells, Cellbarcode:In read1, the bases 1-10,42-51;UMI:In read1, the bases 34-41. The percentage of uniquely mapping reads for genomes of each species with over 85% UMIs assigned to one species was regarded as species-specific cells, while the remaining cells were labeled collisions. Count matrices of bulk RNA-seq of axolotl specimens were generated by featureCounts with default parameters.
For bulk RNA-seq, reads in bam file were aligned to the  genome assembly using STAR version (2.5.2a)  with default configurations. Next, merge the STAR alignment tagged bam were treated using DEseq2 with default configurationsand the reads are annotated with exon tags. Final featurecounts text files were used for downstream analysis.
Assembly: AmexG_v3.0.0(Axolotl genome assembly v3.0.0)(https://genome.axolotl-omics.org/cgi-bin/hgTracks?db=ambMex3&lastVirtModeType=default&lastVirtModeExtraState=&virtModeType=default&virtMode=0&nonVirtPosition=&position=AMEXG_0030000001%3A1000%2D2000&hgsid=43238_8SJNnizHOy6T42X4YaUpQyigV8RT)or(https://www.axolotl-omics.org/assemblies)
Supplementary files format and content: tab-delimited text files of digital expression matrix (dge)(single-cell RNA-seq)

molecule: polyA RNA

organism: Ambystoma mexicanum

extract_protocol:
Total RNA of each tissue was purified using standard TRIzol based protocols for bulk RNA-seq;For single-cell RNA-seq, frozen cells of each tissue were thawed at 37 °C and centrifuged at 500×g for 5 min first. Then, fresh fixed or thawed frozen cells were resuspended with 100 µl PBS, and 400 µl Triton X-100 in PBS (10 µl 10% Triton X-100 in 390 µl PBS) was added. The mixture was incubated on ice for 3 min for permeabilization. Next, the cells were pelleted and resuspended with 500 µl DEPC-treated water, and 3 ml 0.1 N HCl was added slowly. After 5 min of incubation on ice, 3.5 ml Triton X-100 in Tris-HCl (35 µl 10% Triton X-100 in 3.5 ml 1 M Tris-HCl, pH 8.0) was added to quench permeabilization. Cells were then washed once with 1 ml of PBS and filtered with a 40 μm strainer. Cell density was determined using a blood cell counting chamber.
For bulk RNA-seq, RNA extraction followed reverse transcription, second strand DNA synthesis, transposase tagmentation and standard Illumina Nextera library construction. For single-cell RNA-seq, cell numbers for each tissue were estimated, and the corresponding volume of cells in PBS was split into four 96-well plates. For each well, 6.5 µl cells (from 5000 to 20000) was mixed with 0.5 µl 10 mM dNTPs (Thermo Fisher Scientific) and 0.5 µl 100 μM RT barcode primer (see Table S1). The mixture was incubated at 65 °C for 5 min and immediately placed on ice. Two microliters of 5X RT buffer (31 mM Tris-HCl, pH 8.0, 37.5 mM NaCl, 3.1 mM MgCl2, 10 mM DTT), 0.5 µl Maxima H Minus RTase (Thermo Fisher Scientific) and 0.1 µl Murine RNase inhibitor were premixed, and 2.5 µl RT mixture was added to each well. Reverse transcription was carried out by incubating the plates at gradient temperature: 3 cycles (8℃ for 12 s, 15℃ for 45 s, 20℃ for 45 s, 30℃ for 30 s, 42℃ for 2 min, and 55℃ for 3 min) and 55℃ for 60 min. After reverse transcription, plates were placed on ice for 1 min to stop the reaction. All reagents were pooled together into a 15 ml centrifuge tube and then centrifuged at 500 g for 5 min. Pellet was washed with PBS twice and resuspended with hybridization buffer (50 mM Tris-HCl, 10 mM MgCl2, 10 mM DTT, 0.1% Triton X-100, 10% PEG8000 (Sigma-Aldrich), 1% Murine RNase inhibitor). Hybridization buffer within cells was split into eight 96-well plates (3 µl for each well) and 2 µl 25 μM pre-annealing hybridization primers (50 μM HY head oligo, 50μM barcoded HY primer oligo, mixed equally and incubated at 95℃ for 2 min, then slowly cooled to 25℃ with a temperature ramp of −0.1℃/s) were added to each well (see in Table S1). Plates were incubated at 37 °C for 90 min, and 0.5 µl of 100 μM block tail primer oligo was added to block any redundant hybridization primers. After blocking for 30 min at 37 °C, all reagents were pooled into a 15 ml centrifuge tube and then centrifuged at 500 g for 5 min. The pellet was washed with PBS twice and resuspended in 40 µl of PBS. Then, 60 µl PNK mix (10 µl 10X PNK buffer (NEB), 20 µl T4 polynucleotide kinase (NEB), 10 µl 10 mM ATP (NEB), and 20 µl DEPC-treated water) was added. The PNK reaction was carried out at 37℃ for 30 min. Then, 1 ml ice-cold PBS was added to stop the reaction. Cells were then filtered using a 40μm strainer. The reagent was centrifuged at 500 g for 5 min after discarding the supernatants. The pellet was again resuspended in PBS, and the density of cells was estimated. After splitting into one or more 96-well plates. There were 5000~8000 cells per well. The volume was adjusted to 8 µl. Second strand synthesis mix (1.33 µl 10X buffer, 0.66 µl second strand synthesis enzyme mix) was added into each well and the plates were incubated at 16℃ for 3 hours (stop point at 4℃). Then, 10 µl cell lysis buffer (20 mM Tris pH 8.0, 400 mM NaCl, 100 mM EDTA, 4.4% SDS (Sangon Biotech)) and 2 µl proteinase K (Sangon Biotech) were added to the wells. Cell lysis was performed at 55℃ for 60 min. Then, 1 µl PMSF (1 mM) was added to quench the lysis reaction. The plates were incubated at 37℃ for 10 min. For each well, the mixture was then purified using 1.5X VAHTS DNA Clean Beads (Vazyme Biotech). Then, 10 µl of product in DEPC-treated water was transferred into new 96-well plates. For each plate, we randomly chose 10 wells to quantify the dsDNA concentration using Equalbit3.0. Tn5 transposase from the TruePrep DNA Library Prep Kit V2 (Vazyme Biotech) was used for cDNA tagmentation. Then, 10 µl of product in DEPC-treated water was transferred into a new 96-well plate. For each well, 14 µl PCR mix (12 µl 2X KAPA HiFi HotStart ReadyMix (Kapa Biosystems), 1 µl 10 mM P5 primer, and 1 µl 10 mM indexed P7 primer, see Table S1) was added to the plates. The PCR program was as follows: 72 °C for 5 min; 98 °C for 30 s; 12 cycles of 98 °C for 10 s, 60 °C for 30 s, and 72 °C for 1 min; 72 °C for 5 min; and a 4 °C hold. After pooling PCR products, two rounds of size selection with VAHTS DNA clean beads (Vazyme Biotech) were used to purify the DNA library between 300 and 500 bp. The libraries were finally mixed, and the concentration of dsDNA was quantified.The purified linear DNA library was circularized into single strand DNA (ssDNA) library using VAHTS® Circularization Kit for MGI (Vazyme). Then ssDNA library was amplified using DNBSEQ DNB preparation kit (MGI). Amplified DNA nanoball (DNB) was sequenced with custom primers on MGI DNBSQ-T7 with dark reaction model ( Single-cell RNA-seq: 100 cycles of read1 with dark reaction from 11-33, 100 cycles of read2;For NEO1,NEO2: PE150, no dark  reaction). Human and mouse cell line libraries were sequenced with custom primers on Illumina Hiseq Xten with pair-end 150bp mode.

tag: strain

value: d/d

tag: age

value: Adult (1year)

tag: tissue

value: bladder, brain, cloaca, eye, fore limb, hind limb, heart, intestine, kidney, liver, lung, skin, spleen, stomach, tail, gonad


title: Single-cell RNA-seq dataset of metamorphosed axolotl (Batch1), META1, sublibrary 17


title: Single-cell RNA-seq dataset of metamorphosed axolotl (Batch1), META1, sublibrary 19


title: iPSC neurons, GPNMB homozygous knockout, day 14 of differentiation, rep4

value: GPNMB homozygous knock-out


title: scRNA+TCR-seq of tdTomato- GFP- CD4 T cells from the colonic lamina propria [TCR]

description:
GFP_minus_Tom_minus
2549_GFP_minus_Tom_minus_rep1_2_3_TCR_IGO_11874_19_S18

organism: Mus musculus


title: scRNA+TCR-seq of tdTomato+ GFP+ CD4 T cells from the colonic lamina propria

description:
GFP_plus_Tom_plus
2548_GFP_plus_Tom_plus_rep1_2_3_IGO_11963_8_S6


title: Cytosol non-infected RPF, replicate 2

city: Barcelona

first: Juana

last: Díez

state: Barcelona

address: Carrer del Dr. Aiguader 88

department: Department of Experimental and Health Sciences

country: Spain

institute: Universitat Pompeu Fabra

description:
raw_counts_RPF.tsv
Cyt_11_RPF

data_processing:
Library strategy: Ribo-seq
Since mammalian transcripts usually feature several different splice isoforms that can feature different ORFs, we determined the most prominent mRNA isoform for each gene present in our RNA-seq samples to allow a gene-based analysis. To do so, we removed read-through and NMD transcripts from the Appris principal isoforms annotation (http://ubio.bioinfo.cnio.es/data/appris/) for hg38 (based on GENCODE v24) and quantified all remaining transcripts using kallisto (v0.43.0), selecting the protein coding isoform which on average was expressed highest after sleuth’s (v0.29.0) between-sample normalization of the kallisto TPM values. In case multiple transcripts showed equal expression, they were ranked according to the following criteria: 1. Appris classification scheme ('PRINCIPAL:1', 'PRINCIPAL:2', 'PRINCIPAL:3', 'PRINCIPAL:4', 'PRINCIPAL:5', 'ALTERNATIVE:1', 'ALTERNATIVE:2'), 2. lowest CCDS and 3. lowest ENST ID. Lastly, we created a custom bowtie transcriptome index for our HEK 293T cells for ribosome footprint mapping. Prior to mapping to the custom index, we removed sequencing adaptors and trimmed the ribosome profiling reads using cutadapt (v1.91, settings: -a CTGTAGGCACCATCAAT --quality-base 33 -q 28,28 -m 25 -M 35). All surviving Ribo-seq reads were next mapped to an index of known tRNA, rRNA, snRNA, snoRNA and miRNA genes based on GENCODE v24 via bowtie (v1.2.1) and only non-mapping reads were used for mapping to the custom transcriptome index (bowtie v1.2.1, default settings) and subsequent analyses. Read assignment to A- and P-site as well as footprint counting was performed using riboWaltz, selecting reads of 29-33 nt length and using 14-18 nt as corresponding P-site offsets across all samples.  RNA-sequencing reads were mapped against a combined human (hg38) and CHIKV (KT449801.1) genome using TopHat2 (v2.1.0, settings: --max-multihits 1 --b2-very-sensitive --no-coverage-search --library-type fr-firststrand) and supplying an additional transcriptome index based on GENCODE v24. Reads were subsequently quantified using featureCounts (v1.5.1, settings: -s 2 -Q 1 -p) using our custom HEK 293T transcriptome annotation. All genes with fewer than 5 RPF and 10 RNA-seq reads in either ER or cytosol were discarded before continuing our quantitative differential translation analysis. In addition, since RNA-seq reads mapping to the CHIKV SP region can originate from both the full-length CHIKV RNA (NSP ORF) as well as the subgenomic RNA (SP ORF), we calculated the average coverage across the NSP region to estimate how many reads mapping to the SP region should originate from the full-length (=NSP) isoform, assuming a uniform sequencing coverage. Using these estimates we then re-assigned a discrete number of reads from the SP to the NSP ORF for each sample prior to the differential expression analysis. Quantitative analysis of both RNA-seq and Ribo-seq counts were performed using limma-voom (v3.32.2), where changes in relative translational efficiency caused by CHIKV infection were independently modeled for ER and cytosol via an interaction term (~sequencing_type * infection_status).
Genome_build: hg38
Supplementary_files_format_and_content: The file raw_counts_mRNA.tsv contains raw mRNA read counts, whereas raw_counts_RPF.tsv contains raw ribosome footprint counts. Each column represents one sample in lexicographical order.

source_name: whole cell

growth_protocol: Hek293T cells were grown at 37C and 5% CO2 in DMEM + 10% FBS. When reaching 80% confluence cells were splitted.

extract_protocol:
Total RNA was extracted by a hot-phenol protocol and mRNA isolated using the TruSeq stranded mRNA library prep kit (Illumina). For ribosome profiling lysates were treated with RNAse I and subjected to polysome profiling. RNA was collected from the monosome peak and size-selected using a denaturing urea gel. Ribosomal RNA was removed using the Ribo-Zero Gold rRNA Removal kit (Epicenter).
RPF libraries were prepared using the protocol described in (Ingolia, 2009), (Nedialkova and Leidel, 2015) and (Jungfleisch et al. 2017). For RNA-seq libraries, the TruSeq stranded mRNA library prep kit (Illumina) was used.

treatment_protocol: 12 h mock or 12 h CHIKV infection

value: HEK 293T

value: 12 h mock infection

tag: cellular fraction

value: cytosol

tag: molecule

value: Ribosome footprints


title: Cytosol CHIKV-infected RPF, replicate 4

description:
raw_counts_RPF.tsv
Cyt_4_RPF

value: 12 h CHIKV infection


title: scRNA+TCR-seq of tdTomato+ GFP+ CD4 T cells from the colonic lamina propria [HTO]

description:
GFP_plus_Tom_plus
2548_GFP_plus_Tom_plus_rep1_2_3_HTO_IGO_12065_42_S6


title: WM983 Control

city: Sugar Land

first: Kumaraswamy Naidu

last: Chitrala

state: TX

address: 13850 University Blvd, SAB1 Room No: 214

department: Engineering Technology

institute: College of Technology, University of Houston

data_processing:
Rsubread algorithm
Preprocessed data files from the samples were subject to RNA sequencing data analysis using Rsubread package
Assembly: GRCm38

growth_protocol: B16N murine melanoma cells were maintained in DMEM supplemented with 10% fetal bovine serum (FBS) and antibiotics in humidified atmosphere with 5% CO2 at 37C

extract_protocol: RNA libraries for RNA-seq were prepared using  manufacturer's protocols

treatment_protocol:
Cells were exposed to UV for 2 or 5 weeks prior to collection
Cells were transfected with Orai1-WT-CFP, selected with G418, and FACS sorted. Following, cells were grown as individual clones and then collected

value: Human Melanoma Cells

tag: time

value: Day 0


title: UACC257 Control


title: Single-cell RNA-seq dataset of metamorphosed axolotl (Batch1), META1, sublibrary 16


title: WM983 UV3

value: WT, UV exposed

value: UV exposure

value: Week 2


title: Cytosol CHIKV-infected RPF, replicate 3

description:
raw_counts_RPF.tsv
Cyt_3_RPF


title: scRNA+TCR-seq of tdTomato- GFP- CD4 T cells from the colonic lamina propria

description:
GFP_minus_Tom_minus
2549_GFP_minus_Tom_minus_rep1_2_3_IGO_11963_9_S7

value: CD4 T cells


title: scRNA-seq on CD4 T cells from the mLN of colonized Foxp3CNS1- mice

data_processing:
FASTQ files from sequencing were processed by “cellranger count” v4.0.0 using transcriptome refdata-gex-mm10-2020-A.
The resulting count marices were analyzed in scanpy v1.5.1. For preliminary analysis, only cells with at least 100 captured genes were considered, and only genes captured in at least 10 cells were considered. This resulted in 4,111 cells in the sample “GFP-GF”, 4,146 cells in the sample “GFP-Colonized”, 3,635 cells in the sample “CNS1-GF”, and 5,586 cells in the sample “CNS1-Colonized”.
Then normalization was performed using function scanpy.pp.normalize_total() with parameters exclude_highly_expressed=True, max_fraction=0.02. The subsequent analysis focused only on protein-coding genes, and furthermore ribosomal genes were excluded from the analysis. After that, cells with less than 400 captured genes (227 cells collectively over the four samples) were excluded from the analysis, and genes captured in less than 10 cells (9 genes) were excluded.
For preliminary analysis, cells from all four samples combined were clustered into 16 clusters using function scanpy.tl.louvain() with parameter resolution=1.5. Clusters with high expression of B cell genes (120 cells across all four samples) and CD8 T cell/NK/NKT cell genes (183 cells across the four samples) were identified as likely contamination and excluded from the subsequent main analysis.
In the main analysis, dimensionality reduction was performed using PCA, followed by kNN construction using function scanpy.pp.neighbors() with parameters n_neighbors=30, n_pcs=50, use_rep='X_pca', and data was then visualized with UMAP using function scanpy.tl.umap() with parameter min_dist=0.2 and with tSNE using function sc.tl.tsne() with default parameters. Cells then were clustered using package phenograph with parameter k=30 and otherwise default paramerters, resulting in clusters 0-9.
Genome_build: mm10
Supplementary_files_format_and_content: mln-cd4t-scrnaseq.csv is a scRNA-seq raw count matrix for all four samples combined in the CSV format. mln-cd4t-scrnaseq-cells.csv is a CSV file with metadata about cells.

source_name: CD4 T cells from the mLN

growth_protocol: Germ-free male Foxp3GFP and Foxp3CNS1- mice were colonized with SPF fecal microbiota or kept germ-free.

extract_protocol:
9 days post-colonization, a 50%-50% mix of GFP+ and GFP- CD4 T cells was sorted from pooled mLN of 3 mice per group
Single-cell RNA-Seq of FACS-sorted cell populations was performed on a Chromium instrument (10X Genomics) following the user guide manual for 3′ v3 or 5′ v2 chemistry. In brief, FACS-sorted cells were washed once with PBS containing 0.04% bovine serum albumin (BSA) and resuspended in PBS containing 0.04% BSA to a final concentration of 700–1,200 cells per μl. The viability of cells was above 80%, as confirmed with 0.2% (w/v) Trypan Blue staining (Countess II). Cells were captured in droplets. Following reverse transcription and cell barcoding in droplets, emulsions were broken and cDNA was purified using Dynabeads MyOne SILANE followed by PCR amplification per manual instruction. For the 3’ v3 experiment (Foxp3GFP and Foxp3CNS1- CD4 T cells from germ-free and colonized mice), approximately 5000 cells were targeted for each sample. Final libraries were sequenced on Illumina NovaSeq S4 platform (R1 – 28 cycles, i7 – 8 cycles, R2 – 90 cycles).

tag: microbiota

value: colonized


title: Cytosol non-infected RPF, replicate 1

description:
raw_counts_RPF.tsv
Cyt_9_RPF


title: scRNA+TCR-seq of tdTomato- GFP- CD4 T cells from the colonic lamina propria [HTO]

description:
GFP_minus_Tom_minus
2549_GFP_minus_Tom_minus_rep1_2_3_HTO_IGO_12065_43_S2


title: UACC257 UV3


title: Single-cell RNA-seq dataset of metamorphosed axolotl (Batch1), META1, sublibrary 20


title: iPSC neurons, GPNMB homozygous knockout, day 14 of differentiation, rep1

value: iPSC-N

value: iPSC-derived neurons

value: Doxycycline induced differentiation

value: Day 14


title: iPSC neurons, GPNMB homozygous knockout, day 14 of differentiation, rep3

value: GPNMB homozygous knock-out


title: iPSC neurons, GPNMB homozygous knockout, day 14 of differentiation, rep2


title: Cytosol CHIKV-infected RPF, replicate 2

description:
raw_counts_RPF.tsv
Cyt_2_RPF

value: HEK 293T


title: Single-cell RNA-seq dataset of metamorphosed axolotl (Batch1), META1, sublibrary 15

value: d/d

value: Adult (1year)

value: bladder, brain, cloaca, eye, fore limb, hind limb, heart, intestine, kidney, liver, lung, skin, spleen, stomach, tail, gonad


title: Cytosol CHIKV-infected RPF, replicate 1

description:
raw_counts_RPF.tsv
Cyt_1_RPF

value: 12 h CHIKV infection

value: cytosol

value: Ribosome footprints


title: scRNA-seq on CD4 T cells from the mLN of colonized Foxp3GFP mice


title: scRNA-seq on CD4 T cells from the mLN of germ-free Foxp3CNS1- mice

value: germ-free


title: ER non-infected RPF, replicate 3

description:
raw_counts_RPF.tsv
ER_12_RPF

value: 12 h mock infection

value: ER


title: iPSC neurons, GPNMB heterozygous knockout, day 14 of differentiation, rep4

value: GPNMB heterozygous knock-out


title: Single-cell RNA-seq dataset of metamorphosed axolotl (Batch1), META1, sublibrary 13


title: Single-cell RNA-seq dataset of metamorphosed axolotl (Batch1), META1, sublibrary 11


title: iPSC neurons, GPNMB heterozygous knockout, day 14 of differentiation, rep5


title: Single-cell RNA-seq dataset of metamorphosed axolotl (Batch1), META1, sublibrary 10


title: Single-cell RNA-seq dataset of metamorphosed axolotl (Batch1), META1, sublibrary 12


title: FoxoH4

city: Calgary

first: Savraj

last: Grewal

state: Alberta

address: 3330 Hospital Drive

country: Canada

institute: University of Calgary

data_processing:
1.Using Fast QC for reads quality control. The reads number per sample is around 20 million, and average reads length is 51 bp. All samples passed reads quality control.
2. Map the reads to Drosophila_melanogaster: refSeq mRNA (release: 2019-Oct-15) corresponding to dm6 annotation
3. QC for reads mapping quality:  at least 90% of reads pseudo aligned to reference.
4. PCA plot for experiments QC, didn’t see obvious experimental problem, for example, uneven rRNA depletion.
5. Transcripts were quantified using Kallisto (BRAY et al. 2016) referencing refSeq mRNA (release: 2019-Oct-15) corresponding to dm6 annotation. Differential expression testing was performed using sleuth (PIMENTEL et al. 2017).
Assembly: Drosophila_melanogaster refSeq mRNA (release: 2019-Oct-15) corresponding to dm6 annotation
Supplementary files format and content: *hypoxia regulated genes: list of genes that showing significiant changes upon hypoxia exposure.  Fold change indicates differences in expression in hypoxia vs normoxia

organism: Drosophila melanogaster

source_name: whole-body RNA, adult female

extract_protocol:
Adult flies were snap frozen on dry ice in groups of 5. Total RNA was then isolated using Trizol according to the manufacturer’s instructions (Invitrogen; 15596-018). Extracted RNA was then DNase treated (Ambion; 2238G) to be used for subsequent qPCR or mRNA sequencing.
RNA libraries were prepared for sequencing using the Ultra II Directional RNA library kit (New England BioLabs) according the manufacturer's instructions

treatment_protocol: Mated adult female flies (~7 days old, w1118 or foxo mutant) were either maintained in normoxia (ambient air) or exposed to hypoxia for 16hrs by placing them into an airtight glass chamber into which a mix of 1% oxygen/99% nitrogen gas continually flowed. Flow rate was controlled using an Aalborg model P gas flow meter.

value: adult female whole body

tag: Sex

value: female

value: ~7 days

value: foxo

tag: treatment condition

value: hypoxia


title: FoxoH3


title: iPSC neurons, GPNMB heterozygous knockout, day 14 of differentiation, rep3

value: iPSC-N

value: iPSC-derived neurons

value: Doxycycline induced differentiation

value: Day 14


title: ER non-infected RPF, replicate 1

description:
raw_counts_RPF.tsv
ER_9_RPF

value: HEK 293T


title: ER non-infected RPF, replicate 2

description:
raw_counts_RPF.tsv
ER_11_RPF


title: FoxoH2


title: Single-cell RNA-seq dataset of metamorphosed axolotl (Batch1), META1, sublibrary 9

value: d/d

value: Adult (1year)

value: bladder, brain, cloaca, eye, fore limb, hind limb, heart, intestine, kidney, liver, lung, skin, spleen, stomach, tail, gonad


title: iPSC neurons, wild type, day 0 of differentiation, rep5

value: WT

value: Day 0


title: ER CHIKV-infected RPF, replicate 3

description:
raw_counts_RPF.tsv
ER_3_RPF

value: 12 h CHIKV infection

value: ER

value: Ribosome footprints


title: iPSC neurons, GPNMB heterozygous knockout, day 14 of differentiation, rep1

value: GPNMB heterozygous knock-out


title: Single-cell RNA-seq dataset of metamorphosed axolotl (Batch1), META1, sublibrary 14


title: ER CHIKV-infected RPF, replicate 4

description:
raw_counts_RPF.tsv
ER_4_RPF

value: 13 h CHIKV infection


title: FoxoH1

value: adult female whole body

value: female

value: ~7 days

value: foxo


title: ER CHIKV-infected RPF, replicate 2

description:
raw_counts_RPF.tsv
ER_2_RPF


title: FoxoN4

value: normoxia


title: Single-cell RNA-seq dataset of metamorphosed axolotl (Batch1), META1, sublibrary 7


title: scRNA-seq on CD4 T cells from the mLN of germ-free Foxp3GFP mice

value: CD4 T cells

value: germ-free


title: iPSC neurons, wild type, day 0 of differentiation, rep3

value: iPSC-N

value: iPSC-derived neurons

value: Doxycycline induced differentiation


title: iPSC neurons, wild type, day 0 of differentiation, rep4

value: WT


title: Single-cell RNA-seq dataset of metamorphosed axolotl (Batch1), META1, sublibrary 8

value: d/d

value: Adult (1year)

value: bladder, brain, cloaca, eye, fore limb, hind limb, heart, intestine, kidney, liver, lung, skin, spleen, stomach, tail, gonad


title: iPSC neurons, GPNMB heterozygous knockout, day 14 of differentiation, rep2

value: Day 14


title: ER CHIKV-infected RPF, replicate 1

description:
raw_counts_RPF.tsv
ER_1_RPF

value: HEK 293T

value: 12 h CHIKV infection

value: ER

value: Ribosome footprints


title: FoxoN2

value: adult female whole body

value: female

value: ~7 days


title: FoxoN3

value: foxo

value: normoxia


title: iPSC neurons, wild type, day 0 of differentiation, rep2

value: iPSC-N

value: Day 0


title: iPSC neurons, wild type, day 0 of differentiation, rep1

value: iPSC-derived neurons

value: Doxycycline induced differentiation


title: Cytosol non-infected mRNA, replicate 3

description:
raw_counts_mRNA.tsv
Cyt_12_RNA

data_processing:
Since mammalian transcripts usually feature several different splice isoforms that can feature different ORFs, we determined the most prominent mRNA isoform for each gene present in our RNA-seq samples to allow a gene-based analysis. To do so, we removed read-through and NMD transcripts from the Appris principal isoforms annotation (http://ubio.bioinfo.cnio.es/data/appris/) for hg38 (based on GENCODE v24) and quantified all remaining transcripts using kallisto (v0.43.0), selecting the protein coding isoform which on average was expressed highest after sleuth’s (v0.29.0) between-sample normalization of the kallisto TPM values. In case multiple transcripts showed equal expression, they were ranked according to the following criteria: 1. Appris classification scheme ('PRINCIPAL:1', 'PRINCIPAL:2', 'PRINCIPAL:3', 'PRINCIPAL:4', 'PRINCIPAL:5', 'ALTERNATIVE:1', 'ALTERNATIVE:2'), 2. lowest CCDS and 3. lowest ENST ID. Lastly, we created a custom bowtie transcriptome index for our HEK 293T cells for ribosome footprint mapping. Prior to mapping to the custom index, we removed sequencing adaptors and trimmed the ribosome profiling reads using cutadapt (v1.91, settings: -a CTGTAGGCACCATCAAT --quality-base 33 -q 28,28 -m 25 -M 35). All surviving Ribo-seq reads were next mapped to an index of known tRNA, rRNA, snRNA, snoRNA and miRNA genes based on GENCODE v24 via bowtie (v1.2.1) and only non-mapping reads were used for mapping to the custom transcriptome index (bowtie v1.2.1, default settings) and subsequent analyses. Read assignment to A- and P-site as well as footprint counting was performed using riboWaltz, selecting reads of 29-33 nt length and using 14-18 nt as corresponding P-site offsets across all samples.  RNA-sequencing reads were mapped against a combined human (hg38) and CHIKV (KT449801.1) genome using TopHat2 (v2.1.0, settings: --max-multihits 1 --b2-very-sensitive --no-coverage-search --library-type fr-firststrand) and supplying an additional transcriptome index based on GENCODE v24. Reads were subsequently quantified using featureCounts (v1.5.1, settings: -s 2 -Q 1 -p) using our custom HEK 293T transcriptome annotation. All genes with fewer than 5 RPF and 10 RNA-seq reads in either ER or cytosol were discarded before continuing our quantitative differential translation analysis. In addition, since RNA-seq reads mapping to the CHIKV SP region can originate from both the full-length CHIKV RNA (NSP ORF) as well as the subgenomic RNA (SP ORF), we calculated the average coverage across the NSP region to estimate how many reads mapping to the SP region should originate from the full-length (=NSP) isoform, assuming a uniform sequencing coverage. Using these estimates we then re-assigned a discrete number of reads from the SP to the NSP ORF for each sample prior to the differential expression analysis. Quantitative analysis of both RNA-seq and Ribo-seq counts were performed using limma-voom (v3.32.2), where changes in relative translational efficiency caused by CHIKV infection were independently modeled for ER and cytosol via an interaction term (~sequencing_type * infection_status).
Genome_build: hg38
Supplementary_files_format_and_content: The file raw_counts_mRNA.tsv contains raw mRNA read counts, whereas raw_counts_RPF.tsv contains raw ribosome footprint counts. Each column represents one sample in lexicographical order.

value: 12 h mock infection

value: cytosol

value: total RNA


title: Cytosol non-infected mRNA, replicate 2

description:
raw_counts_mRNA.tsv
Cyt_11_RNA


title: Single-cell RNA-seq dataset of metamorphosed axolotl (Batch1), META1, sublibrary 5

value: d/d

value: Adult (1year)

value: bladder, brain, cloaca, eye, fore limb, hind limb, heart, intestine, kidney, liver, lung, skin, spleen, stomach, tail, gonad


title: FoxoN1


title: w1118H4

value: hypoxia


title: Single-cell RNA-seq dataset of metamorphosed axolotl (Batch1), META1, sublibrary 4


title: w1118H3


title: iPSC neurons, GPNMB homozygous knockout, day 0 of differentiation, rep4

value: GPNMB homozygous knock-out


title: Cytosol CHIKV-infected mRNA, replicate 4

description:
raw_counts_mRNA.tsv
Cyt_4_RNA

value: HEK 293T

value: 12 h CHIKV infection


title: Single-cell RNA-seq dataset of metamorphosed axolotl (Batch1), META1, sublibrary 2


title: Single-cell RNA-seq dataset of metamorphosed axolotl (Batch1), META1, sublibrary 3


title: Cytosol CHIKV-infected mRNA, replicate 3

description:
raw_counts_mRNA.tsv
Cyt_3_RNA


title: iPSC neurons, GPNMB homozygous knockout, day 0 of differentiation, rep3


title: iPSC neurons, GPNMB homozygous knockout, day 0 of differentiation, rep2


title: w1118H2

value: adult female whole body

value: female

value: ~7 days


title: Cytosol CHIKV-infected mRNA, replicate 2

description:
raw_counts_mRNA.tsv
Cyt_2_RNA


title: w1118H1


title: w1118N4

value: normoxia


title: Single-cell RNA-seq dataset of metamorphosed axolotl (Batch1), META1, sublibrary 6


title: iPSC neurons, GPNMB homozygous knockout, day 0 of differentiation, rep1

value: iPSC-N

value: Day 0


title: Cytosol CHIKV-infected mRNA, replicate 1

description:
raw_counts_mRNA.tsv
Cyt_1_RNA


title: Single-cell RNA-seq dataset of neotenic axolotl (Batch4), NEO4, sublibrary 94

value: brain, bladder, cloaca, eye, fore limb, hind limb, gill, gonad, pancreas, prostate, heart, intestine, kidney, liver, lung, skin, spleen, stomach, tail


title: Cytosol non-infected mRNA, replicate 1

description:
raw_counts_mRNA.tsv
Cyt_9_RNA

value: 12 h mock infection


title: iPSC neurons, GPNMB homozygous knockout, day 0 of differentiation, rep5

value: iPSC-derived neurons

value: Doxycycline induced differentiation


title: iPSC neurons, GPNMB heterozygous knockout, day 0 of differentiation, rep5

value: GPNMB heterozygous knock-out


title: ER non-infected mRNA, replicate 3

description:
raw_counts_mRNA.tsv
ER_12_RNA

value: ER

value: total RNA


title: Single-cell RNA-seq dataset of neotenic axolotl (Batch4), NEO4, sublibrary 93

value: d/d

value: Adult (1year)


title: ER non-infected mRNA, replicate 2

description:
raw_counts_mRNA.tsv
ER_11_RNA

value: HEK 293T


title: w1118N3


title: Single-cell RNA-seq dataset of neotenic axolotl (Batch4), NEO4, sublibrary 91


title: w1118N2


title: iPSC neurons, GPNMB heterozygous knockout, day 0 of differentiation, rep3


title: Single-cell RNA-seq dataset of neotenic axolotl (Batch4), NEO4, sublibrary 92


title: ER non-infected mRNA, replicate 1

description:
raw_counts_mRNA.tsv
ER_9_RNA


title: iPSC neurons, GPNMB heterozygous knockout, day 0 of differentiation, rep2


title: hisrfp_NC14later_rep_D

city: Madison

first: Melissa

middle: M

last: Harrison

email: mharrison3@wisc.edu, ykaur@wisc.edu, torreszelada@wisc.edu, mmfreund2@wisc.edu

state: WI

address: 420 Henry Mall

department: Biomolecular Chemistry

institute: University of Wisconsin Madison

data_processing:
Raw reads were aligned to the BDGP D. melanogaster genome release 6 (dm6) using hisat2 v2.1.0 with the following parameters: -k 2 –very-sensitive (Kim et al., 2015).
Reads aligning to multiple locations were discarded. SAMtools was used to filter (-q 30) and convert file formats (Li et al. 2009).
Reads were assigned to annotated genes using featureCounts v1.5.3 (Liao et al., 2014) using default parameters and the UCSC annotation (r6.20).
The resultant table of read counts was imported into R v4.1.0 and differential expression was determined using DESeq2 v1.33.5. (Love et al., 2014). Genes with < 50 reads across all samples were filtered out.
Differential expression (adjusted p value < 0.05 and fold change > 1) was determined using the standard DESeq2 analysis.
Genome_build: dm6
Supplementary_files_format_and_content: A combined count table of read counts from each file is provided

source_name: embryo

growth_protocol: standard fly food at 25°C

extract_protocol:
At the appropriate time oocytes and embryos were picked into Trizol (Invitrogen #15596026) with 200 ug/ml glycogen (Invitrogen #10814010) and pierced with a 27G needle to release the RNA.
TruSeq RNA sample prep kit v2

tag: Stage

value: Later Nuclear Cycle 14 embryo


title: hisrfp_NC14later_rep_B


title: Single-cell RNA-seq dataset of neotenic axolotl (Batch4), NEO4, sublibrary 90


title: hisrfp_NC14later_rep_C


title: Single-cell RNA-seq dataset of metamorphosed axolotl (Batch1), META1, sublibrary 1

value: bladder, brain, cloaca, eye, fore limb, hind limb, heart, intestine, kidney, liver, lung, skin, spleen, stomach, tail, gonad


title: iPSC neurons, GPNMB heterozygous knockout, day 0 of differentiation, rep1


title: Single-cell RNA-seq dataset of neotenic axolotl (Batch4), NEO4, sublibrary 95


title: iPSC neurons, GPNMB heterozygous knockout, day 0 of differentiation, rep4


title: ER CHIKV-infected mRNA, replicate 4

description:
raw_counts_mRNA.tsv
ER_4_RNA

value: 13 h CHIKV infection


title: Single-cell RNA-seq dataset of neotenic axolotl (Batch4), NEO4, sublibrary 87


title: hisrfp_NC14early_rep_C

value: Early Nuclear Cycle 14 embryo


title: Single-cell RNA-seq dataset of neotenic axolotl (Batch4), NEO4, sublibrary 88


title: ER CHIKV-infected mRNA, replicate 3

description:
raw_counts_mRNA.tsv
ER_3_RNA

value: 12 h CHIKV infection


title: hisrfp_NC14later_rep_A


title: ER CHIKV-infected mRNA, replicate 2

description:
raw_counts_mRNA.tsv
ER_2_RNA


title: Single-cell RNA-seq dataset of neotenic axolotl (Batch4), NEO4, sublibrary 89


title: ER CHIKV-infected mRNA, replicate 1

description:
raw_counts_mRNA.tsv
ER_1_RNA


title: SI_T_8_3145_SI_T

city: San Diego

first: Gooyoung

last: Seo

state: CA

address: 9420 Athena Cir, La Jolla

institute: La Jolla Institute for immunology

data_processing:
Reads passing Illumina filters were further filtered for those mapping to tRNA, rRNA, adapters, and spike-in controls using Bowtie v1.0.0 (-S --fr)
Filtered reads were mapped against the mouse mm10 reference genome using TopHat (v 1.4.1)
Reads mapping to the reference genome are further filtered to remove low-complexity reads with PRINSEQ v0.20.3 (-lc_method "dust" -lc_threshold 4)
Sequencing read coverage per gene was counted using htseq-count program (v 0.6.0) and features with 0 counts across all samples were removed
Assembly: mm10
Supplementary files format and content: count matrix with rows as genes and columns as samples

source_name: small intestine

extract_protocol:
RNA-seq libraries were prepared from sorted proximal SI IEC or from sorted SI TCRab+CD8aa+ IET using the SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian (TaKaRa). Libraries were sequenced on an Illumina HiSeq 2500, generating 50 bp single-end reads.
RNA libraries were prepared for sequencing using standard Illumina protocols

value: intraepithelial lymphocytes, TCRab+CD8aa+ IET

value: Villin Cre x Hvemfl/fl, Hvem[delta]IEC mice


title: hisrfp_NC14early_rep_D


title: SI_T_9_3146_SI_T

value: Hvemfl/fl mice


title: hisrfp_NC13_rep_D

value: Nuclear Cycle 13 embryo


title: Single-cell RNA-seq dataset of neotenic axolotl (Batch4), NEO4, sublibrary 85

value: brain, bladder, cloaca, eye, fore limb, hind limb, gill, gonad, pancreas, prostate, heart, intestine, kidney, liver, lung, skin, spleen, stomach, tail


title: hisrfp_NC14early_rep_B


title: hisrfp_NC14early_rep_A


title: SI_T_7_3142_SI_T


title: Single-cell RNA-seq dataset of neotenic axolotl (Batch4), NEO4, sublibrary 83


title: Single-cell RNA-seq dataset of neotenic axolotl (Batch4), NEO4, sublibrary 84


title: Single-cell RNA-seq dataset of neotenic axolotl (Batch4), NEO4, sublibrary 82


title: SI_T_11_3148_SI_T


title: hisrfp_NC13_rep_A


title: hisrfp_NC13_rep_C


title: hisrfp_NC12_rep_D

value: Nuclear Cycle 12 embryo


title: SI_T_10_3147_SI_T


title: 3148_P_EC

source_name: proximal small intestine

value: intestinal epithelial cells


title: Single-cell RNA-seq dataset of neotenic axolotl (Batch4), NEO4, sublibrary 86


title: hisrfp_NC12_rep_C


title: hisrfp_NC12_rep_B


title: 3146_P_EC


title: ISL1CKO_P9_H3K4me3_1

city: Vestec

first: Šárka

last: Benešová

address: Prumyslova 595

department: Institute of Biotechnology

country: Czech Republic

institute: Czech Academy of Science

library_source: genomic

data_processing:
Sequencing data were mapped using bowtie2 (version 2.2.5) to mouse genome GRCm38 primary assembly. PCR duplicates were not removed.
Reads were filtered for those with quality mapping higher than 2 with samtools (version 1.3.1).
Only fragments with mapped pairs on the same chromosome and fragment length lower than 1000 bp were kept.
Peaks were called with SEACR 1.3. using stringent mode and edevelopmental stage relevant IgG control sample as background.
Assembly: GRCm38.p6 (Ensembl)
Supplementary files format and content: bigWig files generated from bam files using bamCoverage from deeptools (version 3.5.1), CPM (counts per milion) normalized.
Library strategy: CUT&Tag

molecule: genomic DNA

source_name: Pancreas

growth_protocol: Cre/loxP system was used to delete ISL1 from Neurod1+ pancreatic cells

extract_protocol:
The pancreata microdissected from E14.5 embryos were directly trypsinized and prepared for FACS. Briefly, tissue was dissociated using 0.05% trypsin/0.53 mM EDTA at 37oC for 5 min. Digestion was stop by the FACS buffer (2% FBS and 10 mM EGTA in PBS 46), and cells were then 1x washed by FACS buffer. The pancreata from P9-P10 pups were first perfused by 1mg/ml collagenase in Hank’s balanced salt solution, dissected, and incubated at 37oC for 10-12 min to release endocrine cells/islets. Digested pancreatic tissue was washed 3x by 1% FBS in Hank’s solution. To generate single cells, the tissue was further dissociated by trypsinization as described above. Finally, cell suspensions were filtered through 40 μm nylon mesh and immediately tdTomato+ cells were sorted using a flow cytometer (BD FACSAriaTM Fusion), through a 100 μm nozzle in 20 psi, operated with BD FACSDiva™ Software. Freshly FACS-sorted tdTomato+ pancreatic endocrine cells were captured by Concanavalin A-coated magnetic beads to facilitate subsequent washing steps. CUT&Tag validated primary antibodies anti-H3K4me3, anti-H3K27me3, normal rabbit IgG negative control, and anti-rabbit secondary antibody were used. Binding of pAG-Tn5 (EpiCypher,15-1017, 2.5μL/reaction) was at RT for 60 min followed by tagmentation. To stop tagmentation and solubilize DNA fragments, 1.67 μL 0.5M EDTA, 0.5 μL 10% SDS and 0.42 μL 20 mg/mL Proteinase K (20mg/mL) was added to each sample. Samples were incubated for 1 hr at 55 ºC to digest (and reverse cross-links), followed by DNA precipitation and purification. DNA was dissolved in 22 μL of 1 mM Tris-HCl pH 8, 0.1 mM EDTA buffer and utilized as template for library generation with Universal i5 Primer and Uniquely Barcoded i7 Primers for Illumina.
21 μL DNA + 2 μL of 10 μM Universal or barcoded i5 primer + 2 μL of 10 μM uniquely barcoded i7 primer (using a different barcode for each sample) and 25 μL non-hot start CUTANA® High Fidelity 2x PCR Master Mix (EpiCypher, 15-1017) was added to each thin-wall 0.2 ml PCR tube. Library was amplified under CUT&Tag-specific PCR cycling parameters: a)5 min at 58°C; b) 5 min at 72°C; c) 45 sec at 98°C; d) 15 sec at 98°C; e) 10 sec at 60°C; f) 1 min 72°C. Steps "d-e" were repeated 26 or 27 times. DNA cleanup was performed using 1.3x AMPure beads (Beckman Coulter, A63880). Each purified PCR DNA library for Illumina sequencing was tested on Agilent High Sensitivity DNA Chip. MiSeqReagent Kit v3 (150-cycle) was used for sequencing.

value: Pancreas

tag: developmental stage

value: endocrine cells

value: Isl1 knockout

tag: cut&tag validated_antibody


title: 3145_P_EC

tag: cell type


title: ISL1CKO_P9_H3K27me3_3

tag: genotype


title: ISL1CKO_P9_H3K4me3_2


title: hisrfp_NC12_rep_A


title: 3147_P_EC


title: ISL1CKO_P9_H3K27me3_2


title: SI_T_6_3141_SI_T


title: ISL1CKO_P9_H3K27me3_1


title: 3142_P_EC


title: ISL1CKO_P10_H3K4me3_1


title: ISL1CKO_E14.5_H3K4me3_2


title: hisrfp_NC10_rep_C

value: Nuclear Cycle 10 embryo


title: hisrfp_NC10_rep_B


title: SI_T_1_3134_SI_T


title: ISL1CKO_E14.5_H3k4me3_1


title: hisrfp_NC10_rep_A


title: 3134_P_EC


title: hisrfp_oocyte_rep_D

source_name: oocyte

value: late stage oocyte


title: HEK293T alt-LAMA3 KO 3

first: ZHENKUN

address: Fucheng Road No.1

institute: Hangzhou Institute of Medicine, CAS

data_processing:
Ruddle cluster from Yale University
Sequence reads were trimmed for adaptor sequence/low-quality sequence using cutadptor
Trimmed sequence reads were mapped to Hg38 using STAR and Salmon
Assembly: hg38
Supplementary files format and content: tab-delimited text files from Salmon

growth_protocol: HEK293T and alt-LAMA3 KO cells were maintained in DMEM supplemented with 10% fetal bovine serum (FBS) and antibiotics in humidified atmosphere with 5% CO2 at 37C

extract_protocol:
RNA was harvested using Rneasy mini plus kit (Qiagen). 1.3 ug of total RNA was used for the construction of sequencing libraries.
RNA libraries for RNA-seq were prepared using SMARTER mRNA-Seq Library Prep Kit following manufacturer's protocols.

tag: cell line

value: Human embryonic kidney 293 cell

value: alt-LAMA3 KO

tag: treatment

value: Day 0


title: HEK293T alt-LAMA3 KO 1


title: HEK293T alt-LAMA3 KO 2


title: ISL1CKO_E14.5_H3K27me3_3


title: ISL1CKO_E14.5_H3K27me3_4


title: hisrfp_NC10_rep_D


title: HEK293T 3

value: WT


title: HEK293T 2


title: HEK293T 1


title: 3141_P_EC


title: YG_sb rep2

city: Nanchang

first: Haihui

last: Fu

address: 1101#, Zhimin Avenue，Nanchang, Jiangxi Province

institute: Jiangxi Agricultural University

data_processing:
Illumina Casava1.7 software used for basecalling.
The quality of Sequenced reads were check, and masked for low-complexity or low-quality sequence, then mapped to mm8 whole genome using bowtie v0.12.2 with parameters -q -p 4 -e 100 -y -a -m 10 --best --strata
Genome_build: Oryza_sativa.IRGSP-1.0.dna.toplevel.fa
Supplementary_files_format_and_content: Matrix table with raw gene counts for every gene and every sample

organism: Oryza sativa

source_name: leaf sheath

growth_protocol: select seeds with consistent buds and sow them in 4*8-hole seedling trays, one plant per pot, and grow in an artificial climate chamber

extract_protocol:
RNA was isolated using Trizol reagent Kit.
RNA libraries were prepared for sequencing using standard Illumina protocols

treatment_protocol: Choose 4 leaves stage robust seedlings to inoculate sheath blight, insert 5mm mycelium balls into the inner side of the leaf sheaths of the inverted 2 leaves, and non-inoculation control at the same time.At 18h after inoculation, a total of 1.0 cm samples from the upper and lower part of the inoculated leaf sheath were cut

tag: develomental stage

value: seedling stage

tag: tag

value: sheath blight


title: control_NC14later_rep_D

value: Later Nuclear Cycle 14 embryo

value: brat Df/cyo or brat fs1/cyo


title: hisrfp_oocyte_rep_B


title: hisrfp_oocyte_rep_A


title: hisrfp_oocyte_rep_C


title: ISL1CKO_E14.5_H3K27me3_1


title: ISL1CKO_E14.5_H3K27me3_2


title: control_NC14later_rep_C


title: IgG_control_P9

value: mix of WT and ISL1CKO

value: IgG


title: YG_sb rep1


title: YG_ck rep3

value: non-inoculation control


title: 3138_P_EC

value: intestinal epithelial cells

value: Hvemfl/fl mice


title: IgG_control_E14.5

value: Pancreas

value: endocrine cells


title: Hyper_Ins_Rep3

city: Cambridge

first: Max

last: Friesen

address: 455 Main St

institute: Whitehead Institute

data_processing:
Reads were mapped to the hg38 version of the human genome, containing only canonical chromosomes, with STAR using a GTF file downloaded from ESEMBL version GRCh38.99.  We set the overhang parameter to 50 and the "alignIntronMax" parameter to 95000.
Read counting was done with featureCounts setting the parameter "-p -s 2" and the same GTF file used on the mapping step.
Supplementary files format and content: hg38, gene annotation GRCh38.99
Supplementary files format and content: Text file containing gene counts.

source_name: hESC-differentiated adipocyte

extract_protocol:
RNA is extracted from the cells with TRI reagent, chloroform extracted and isopropanol precipitated
Libraries were prepared using the KAPA mRNA HyperPrep Kit, according to manufacturer’s directions
Libraries are stranded. We obtained 50 nt long paired-end reads.

value: hESC-differentiated adipocyte

value: hESC

tag: procedure

value: hESC-adipocyte differentiation, then 5 days low-glucose DMEM+3nM insulin

value: 10nM insulin for 4 hours

tag: replicate

value: Replicate 3


title: YG_ck rep2


title: Hyper_Ins_Rep2

tag: tissue

value: WT

value: Replicate 2


title: YG_sb rep3

value: seedling stage

value: sheath blight


title: control_NC14later_rep_A

value: Later Nuclear Cycle 14 embryo

value: brat Df/cyo or brat fs1/cyo


title: Control_P9_H3K27me3_2


title: Control_P9_H3K27me3_3


title: Hyper_Ins_Rep1

value: Replicate 1


title: control_NC12_rep_D

value: Nuclear Cycle 12 embryo


title: Control_P9_H3K27me3_1


title: C_sb rep3


title: YG_ck rep1

value: non-inoculation control


title: control_NC12_rep_B


title: C_sb rep1


title: Control_P10_H3K4me3_1


title: C_sb rep2


title: Sensitized_Ins_Rep2

value: hESC-adipocyte differentiation, then 5 days low-glucose DMEM+100pM insulin


title: Sensitized_Ins_Rep1


title: Control_E14.5_H3K4me3_2

value: Pancreas


title: Sensitized_Ins_Rep3


title: Control_E14.5_H3K4me3_1

value: endocrine cells


title: control_NC12_rep_C


title: control_NC12_rep_A


title: Control_E14.5_H3K27me3_4


title: Published_Ins_Rep3

value: hESC-differentiated adipocyte

value: hESC-adipocyte differentiation


title: control_NC14later_rep_B


title: control_NC10_rep_D

value: Nuclear Cycle 10 embryo


title: C_ck rep3


title: R317_Negative

city: Vancouver

first: Francis

middle: C

last: Lynn

state: BC

address: 950 28th Ave W

department: Surgery

institute: University of British Columbia

description: additional donor metrics at https://www.epicore.ualberta.ca/IsletCore/

data_processing:
Illumina bcl files from the NextSeq 500 sequencer  were used as input to demultiplex each sample in each sequencing run and generate fastq files using 10x Genomics Cell Ranger software version 3.0.2. in a linux environment (cellranger mkfastq).
Cell Ranger (cellranger count) was used to take the generated fastq files and align them to the GRCh38 genome, count barcodes, count Unique Molecular Identifiers (UMI), and filter out suspected empty droplets/background noise.
Cell Ranger (cellranger aggr) was used to aggregate all 6 outputs per donor: 3 experimental conditions, 2 sequencing runs' worth of data for each. This generated 1 dataset per donor.
The output matrices of Cell Ranger was used as input into downstream QC and filtering steps on R package Scater. In each donor's dataset, Scater was used to remove cells with fewer than 1000 transcripts, potential debris or multiplets, low-abundance genes, and genes with a high dropout rate.
The output of Scater was used as input into the R package Seurat (version 3). Seurat was used to filter out genes not expressed in at least 3 cells, and cells that did not express more than 1000 genes.
Seurat was used to filter each donor dataset's cells one more time, keeping cells that had greater than 3000 genes (nFeature >3000) and less than 40% mitochondrial genes expressed in a cell.
Each dataset was normalized and scaled using the SCTransform method in the Seurat package.
The 3 donors' datasets were integrated using Seurat, generating a final integrated Seurat Object (.rds file) that was used for downstream dimensional reduction, clustering, visualization, and determination of differentially expressed genes and calcium-regulated genes.
Genome_build: GRCh38
Supplementary_files_format_and_content: Integrated Seurat Object (.rds) of all donors, experimental conditions, and sequencing runs.

source_name: islets of Langerhans

growth_protocol: Human islets were isolated from cadaveric donors at the ADI Islet Core and shipped overnight to the BC Children's Hospital Research Institute in CMRL 1066 media as a pellet. 450 islets per donor were manually picked, and cultured overnight in CMRL 1066 media in 5% CO2 humidified incubator at 37 degrees Celsius.

extract_protocol:
After treatment incubation, all islets in 1 well were spun down at 300g for 3 minutes and supernatant was removed. The pellet was washed with magnesium and calcium-free PBS with 0.5mM EDTA. The cells were spun down once more and the PBS was removed. 50uL of PBS and 200uL of 0.25% trypsin/EDTA was added to the islets and they were incubated for 15 minutes at 37 degrees Celsius. The tube was mildly agitated with a flick every 2-3 minutes to disperse the islet cells. After trypsinization, 250uL of FBS and 1mL of PBS were added to neutralize the trypsin. The cells were spun down, the supernatant was removed, and the dispersed cells were resuspended in 300uL of PBS with 2% FBS for filtering through a 40um filter and counting on a hemacytometer. Once cell numbers were confirmed, the cells were spun down for a final time, the supernant was removed, and the cells were resuspended in a volume of magnesium and calcium-free PBS to achieve a single-cell solution of 1000 cells/uL. The resuspended cell solutions were placed on ice for immediate progression to single-cell library preparation.
The 10x Genomics Chromium Controller was used to capture single cells, with each treatment condition from each donor islet preparation as 1 sample. cDNA libraries were prepared using 10x Genomics Single Cell 3' version 2 reagent kit and workflow for donors R253 and R282, and version 3 reagent kit and workflow for donor R317, according to the manufacturer's instructions.

treatment_protocol: The morning following overnight culture, 150 islets were seeded per well in a 12-well plate for a total of 3 wells. Each well contained KRBH buffer with 1 of 3 experimental conditions: Low) 2.8mM glucose, Positive) 25mM glucose + 40mM KCl, or Negative) 25mM glucose + 40mM KCl + 5mM Egtazic acid. Intact islets were incubated in 1 of these 3 conditions for 1 hour.

value: islets of Langerhans (pancreas)

tag: donor id

value: 25mM glucose, 40mM KCl, 5mM egtazic acid (1 hour)


title: C_ck rep2


title: control_NC10_rep_C


title: Published_Ins_Rep2

value: hESC

value: WT

value: 10nM insulin for 4 hours


title: R317_Positive

value: 25mM glucose, 40mM KCl (1 hour)


title: Published_Ins_Rep1


title: B_sb rep3

value: seedling stage

value: sheath blight


title: Hyper_Rep2

value: hESC-adipocyte differentiation, then 5 days low-glucose DMEM+3nM insulin

value: Replicate 2


title: Hyper_Rep3

value: Replicate 3


title: B_sb rep2


title: control_oocyte_rep_D

value: late stage oocyte

value: brat Df/cyo or brat fs1/cyo


title: B_sb rep1


title: control_NC10_rep_A


title: R253_Negative


title: B_ck rep3

value: non-inoculation control


title: Hyper_Rep1

value: hESC-differentiated adipocyte

value: Replicate 1


title: R282_Positive


title: R282_Negative


title: R282_Low

value: 2.8mM glucose (1 hour)


title: control_oocyte_rep_C


title: control_oocyte_rep_B


title: R253_Positive

value: islets of Langerhans (pancreas)


title: B_ck rep2


title: Sensitized_Rep1

value: hESC-adipocyte differentiation, then 5 days low-glucose DMEM+100pM insulin


title: control_oocyte_rep_A


title: Sensitized_Rep2

value: hESC


title: control_NC10_rep_B

value: Nuclear Cycle 10 embryo


title: R317_Low


title: B_ck rep1


title: C_ck rep1


title: Published_Rep3

value: WT

value: hESC-adipocyte differentiation


title: bratmut_NC14later_rep_D

value: Later Nuclear Cycle 14 embryo

value: brat Df/fs1


title: Sensitized_Rep3


title: A_sb rep3

value: seedling stage

value: sheath blight


title: bratmut_NC14later_rep_C


title: A_sb rep2


title: Published_Rep1


title: A_sb rep1


title: bratmut_NC14later_rep_B


title: bratmut_NC14later_rep_A


title: A_ck rep3


title: bratmut_NC14early_rep_C

value: Early Nuclear Cycle 14 embryo


title: Primary_Rep3

source_name: Adipose

value: Adipose

value: Primary adipocyte

value: Primary cell isolation

value: Replicate 3


title: bratmut_NC14early_rep_D


title: Primary_Rep2

value: Replicate 2


title: A_ck rep2

value: non-inoculation control


title: R253_Low

value: islets of Langerhans (pancreas)

value: 2.8mM glucose (1 hour)


title: bratmut_NC14early_rep_B


title: bratmut_NC14early_rep_A


title: Primary_Rep1

value: Replicate 1


title: K562_dTAG_LIMeID_EZH2degron_rep2

first: Brian

last: Liau

state: MA

address: 12 Oxford St.

department: Department of Chemistry and Chemical Biology

institute: Harvard University

data_processing:
Reads were aligned to the hg38 reference genome containing no ALT contigs utilizing the Biscuit alignment pipeline with the following parameters: “biscuit align -t 16 -R '@RG\tID' Read1.fastq.gz Read2fastq.gz | samblaster | samtools sort -o sample.bam -O BAM –” (Biscuit, version 0.3.16).
To identify both GpC methylation and CpG methylation, the Biscuit pileup program (version 0.3.16) was utilized with the following parameters: -q 12, -N.
To obtain position specific information on GpC and CpG methylation, the Biscuit vcf2bed program was employed with the following parameters:"-k 1" and -t gch” or “-t hcg” to exclude GpCpG sites, in which methylation signal would be ambiguous.
The bedgraph files generated from the methylation analysis were converted to bigwig format utilizing the bedGraphToBigWig function (UCSC).
Assembly: hg38
Supplementary files format and content: Bigwig files for GpC and CpG methylation were generated using Biscuit (version 0.3.16) and the bedGraphToBigWig (UCSC) utility.

source_name: dTAG-EZH2 K562 knock-in expressing Flag-NLS-M.CviPI-Lamin-B1, dTAG-13 72 hours, Doxycycline 24 hours

growth_protocol: K562 cells were grown in RPMI-1640 supplemented with 10% FBS, 100 U/mL penicillin and 100 μg/mL streptomycin.

extract_protocol:
Cell pellets of 1 million cells each were harvested by centrifugation and washed with PBS (Corning). DNA was purified through QIAamp DNA blood mini kit (Qiagen) with the addition of RNAse and sheared to a size of 400 bp using a Covaris sonicator (S220). DNA was subsequently bisulfite converted with the EpiTect Fast Bisulfite Conversion Kit (Qiagen).
LIMe-ID libraries were generated using the Accel-NGS-methyl-Seq Kit (Swift Biosciences) with 9 cycles of amplification.
LIMe-ID

treatment_protocol:
dTAG-EZH2 K562 knock-in Flag-NLS-M.CviPI-Lamin-B1 cells were treated with dTAG-13 (500nM) for 72 hours and induced with doxcycline (1µg/mL) for the final 24 hours of treatment.
For the inhibitor LIMe-ID experiment, cells were seeded at 125,000 cell/mL and then treated with GSK343 (1 µM), EED226 (5 µM), or vehicle DMSO (0.1% v/v) for 72 hours. For the degradation LIMe-ID experiment, cells were seeded at 125,000 cell/mL and then treated with dTAG-13 (500 nM) or vehicle DMSO (0.1% v/v) for 72 hours. For the final 24 hours of the treatment, doxycycline (1µg/mL) was added.

value: chronic myeloid leukemia


title: K562_dTAG_LIMeID_EZH2degron_rep1


title: YZ, H3K9me2, rep2

city: Guangzhou

first: Juntao

last: Wang

state: Guangdong

address: Wushan Street

department: Collage of Horticulture

institute: South China Agricultural University

data_processing:
ChIP-seq reads were trimmed using Trimmomatic 0.38 with the parameter  SLIDINGWINDOW:4:15 LEADING:3 TRAILING:3 ILLUMINACLIP:adapters.fa:2:30:7:1:true MINLEN:36
ChIP-seq reads were aligned to Ca_59 genome using bwa version 0.7.17.
Enrichment peaks were called using MACS2 (2.2.7.1) with default parameters.
The ChIP-seq intensity of a genomic bin (i.e. 10 kb and 40 kb) was calculated using the bamCoverage tool from DeepTools (3.3.0) with the read coverage normalized in CPM.
Assembly: Ca_59/GCA_021292135.1
Supplementary files format and content: bedgraph
Supplementary files format and content: bigWig, narrowPeak

organism: Capsicum annuum

source_name: Young Leaves

extract_protocol:
Grind young leaf (2g) into a fine powder in liquid nitrogen and then crosslinked with 1% formaldehyde for 10 min at room temperature.
After sonication, immunoprecipitation was performed with antibodies. ChIP was performed using antibodies against the following: H3K4me3 (Abcam, ab8580), H3K27me3 (Millipore 07-499), and H3K9me2 (Abcam, ab1220). The immunoprecipitated complex was washed, and DNA was extracted and purified by Universal DNA Purification Kit (QIAquick PCR Purification Kit, 28106). The ChIP-Seq library was prepared using the ChIP-Seq DNA sample preparation kit (NEBNext® Ultra™II DNA) according to the manufacturer’s instructions. For ChIP-seq, extracted DNA was ligated to specific adaptors followed by deep sequencing in the Illumina Novaseq 6000 using 150bp paired-end. Samples were prepared and sequenced with assistance from Shanghai Jiayin Biotechnology Co., Ltd.

tag: cultivar

value: Young Leaves

tag: chip antibody

value: H3K9me2 (Abcam, ab1220)


title: bratmut_NC13_rep_D

value: Nuclear Cycle 13 embryo


title: K562_DMSO_LIMeID_EZH2degron_rep2

source_name: dTAG-EZH2 K562 knock-in expressing Flag-NLS-M.CviPI-Lamin-B1, DMSO 72 hours, Doxycycline 24 hours

treatment_protocol:
dTAG-EZH2 K562 knock-in Flag-NLS-M.CviPI-Lamin-B1 cells were treated with DMSO (0.1%) for 72 hours and induced with doxcycline (1µg/mL) for the final 24 hours of treatment.
For the inhibitor LIMe-ID experiment, cells were seeded at 125,000 cell/mL and then treated with GSK343 (1 µM), EED226 (5 µM), or vehicle DMSO (0.1% v/v) for 72 hours. For the degradation LIMe-ID experiment, cells were seeded at 125,000 cell/mL and then treated with dTAG-13 (500 nM) or vehicle DMSO (0.1% v/v) for 72 hours. For the final 24 hours of the treatment, doxycycline (1µg/mL) was added.


title: Published_Rep2

value: hESC-differentiated adipocyte

value: hESC

value: WT

value: hESC-adipocyte differentiation


title: bratmut_NC13_rep_C

value: brat Df/fs1


title: bratmut_NC13_rep_A


title: K562_DMSO_LIMeID_EZH2degron_rep1


title: YZ, H3K9me2, rep1 input


title: K562_GSK343_LIMeID_rep2

source_name: M.CviPI-Lamin-B1 K562 clonal cells, GSK343 72 hours, Doxycycline 24 hours

treatment_protocol:
M.CviPI-Lamin-B1 K562 clonal cells were treated with GSK343 (1µM) for 72 hours and induced with doxcycline (1µg/mL) for the final 24 hours of treatment.
For the inhibitor LIMe-ID experiment, cells were seeded at 125,000 cell/mL and then treated with GSK343 (1 µM), EED226 (5 µM), or vehicle DMSO (0.1% v/v) for 72 hours. For the degradation LIMe-ID experiment, cells were seeded at 125,000 cell/mL and then treated with dTAG-13 (500 nM) or vehicle DMSO (0.1% v/v) for 72 hours. For the final 24 hours of the treatment, doxycycline (1µg/mL) was added.


title: A_ck rep1

value: seedling stage


title: bratmut_NC12_rep_D

value: Nuclear Cycle 12 embryo


title: YZ, H3K9me2, rep1


title: YZ, H3K4me3, rep2

value: H3K4me3 (Abcam, ab8580)


title: K562_GSK343_LIMeID_rep1


title: YZ, H3K4me3, rep2 input


title: YZ, H3K9me2, rep2 input


title: K562_EED226_LIMeID_rep2

source_name: M.CviPI-Lamin-B1 K562 clonal cells, EED226 72 hours, Doxycycline 24 hours

treatment_protocol:
M.CviPI-Lamin-B1 K562 clonal cells were treated with EED226 (5µM) for 72 hours and induced with doxcycline (1µg/mL) for the final 24 hours of treatment.
For the inhibitor LIMe-ID experiment, cells were seeded at 125,000 cell/mL and then treated with GSK343 (1 µM), EED226 (5 µM), or vehicle DMSO (0.1% v/v) for 72 hours. For the degradation LIMe-ID experiment, cells were seeded at 125,000 cell/mL and then treated with dTAG-13 (500 nM) or vehicle DMSO (0.1% v/v) for 72 hours. For the final 24 hours of the treatment, doxycycline (1µg/mL) was added.


title: bratmut_NC12_rep_C


title: YZ, H3K4me3, rep1 input


title: K562_EED226_LIMeID_rep1


title: YZ, H3K4me3, rep1


title: bratmut_NC12_rep_B


title: K562_DMSO_LIMeID_rep1

source_name: M.CviPI-Lamin-B1 K562 clonal cells, DMSO 72 hours, Doxycycline 24 hours

treatment_protocol:
M.CviPI-Lamin-B1 K562 clonal cells were treated with DMSO (0.1%) for 72 hours
For the inhibitor LIMe-ID experiment, cells were seeded at 125,000 cell/mL and then treated with GSK343 (1 µM), EED226 (5 µM), or vehicle DMSO (0.1% v/v) for 72 hours. For the degradation LIMe-ID experiment, cells were seeded at 125,000 cell/mL and then treated with dTAG-13 (500 nM) or vehicle DMSO (0.1% v/v) for 72 hours. For the final 24 hours of the treatment, doxycycline (1µg/mL) was added.


title: K562_DMSO_LIMeID_rep2


title: K562_GSK343_input_rep2

data_processing:
Reads were aligned to a composite reference genome for dm6 and hg38 utilizing bowtie2 (version 2.3.2) according to the Spiker (version 1.03) analysis framework.
Aligned reads were sorted using samtools (version 0.1.19).
The Spiker split_bam.py script was run to calculate scaling factors and to split reads into an alignment file containing reads specifically aligning to the hg38 genome (Spiker, version 1.0.3).
The alignment file was then converted into spike-in normalized bigwig file for further analysis and visualization utilizing the deepTools (version 3.4.3) bamCoverage command with the following parameters: -binSize 50 --normalizeUsing RPKM --ignoreDuplicates --ignoreForNormalization chrM --extendReads –scaleFactor.
Peaks for individual H3K27me3 replicates were called utilizing MACS2 “callpeak” function with the following parameters: - -f BEDPE -B -g hs --broad --broad-cutoff 0.2. Regions within 10kb of another were merged and individual replicate peaks were combined utilizing Bedtools2 merge.
Assembly: hg38
Supplementary files format and content: BigWig density files were generated by deepTools (version 3.4.3).

source_name: Flag-NLS-M.CviPI-Lamin-B1 K562 clonal cells, EZH2i (GSK343) 72 hours, Doxycycline 24 hours

extract_protocol:
Cells were fixed in 1% formaldehyde, washed, snap frozen, and stored at -80ºC. Sonication of crosslinked cells was calibrated such that DNA was sheared to between 300bp and 2000bp. Drosophila S2 chromatin (Active Motif) and spike-in antibody (Active Motif) were added following lysis and sonication but prior to immunoprecipitation.
ChIP DNA was used to generate sequencing libraries by end repair (End-It DNA repair kit, Epicentre), 3’ A base overhang addition via Klenow fragment (NEB), and ligation of barcoded sequencing adapters.
Quantitative spike-in ChIP-Seq

treatment_protocol:
Flag-NLS-M.CviPI-Lamin-B1 K562 clonal cells were treated with GSK343 (1µM) for 72 hours and induced with doxcycline (1µg/mL) for the final 24 hours of treatment.
Cells were seeded at 125,000 cell/mL and then treated with GSK343 (1 µM) or DMSO (0.1% v/v)) for 72 hours. For the final 24 hours of the treatment, doxycycline (1µg/mL) was added.


title: bratmut_NC10_rep_C

value: Nuclear Cycle 10 embryo


title: YZ, H3K27me3, rep2

value: H3K27me3 (Millipore 07-499)


title: bratmut_NC10_rep_D


title: YZ, H3K27me3, rep2 input


title: bratmut_NC10_rep_B


title: YZ, H3K27me3, rep1


title: bratmut_NC12_rep_A


title: K562_GSK343_input_rep1


title: YZ, H3K27me3, rep1 input


title: bratmut_NC10_rep_A


title: K562_DMSO_input_rep2

source_name: Flag-NLS-M.CviPI-Lamin-B1 K562 clonal cells, DMSO 72 hours, Doxycycline 24 hours

treatment_protocol:
Flag-NLS-M.CviPI-Lamin-B1 K562 clonal cells were treated with DMSO (0.1% v/v) for 72 hours and induced with doxcycline (1µg/mL) for the final 24 hours of treatment.
Cells were seeded at 125,000 cell/mL and then treated with GSK343 (1 µM) or DMSO (0.1% v/v)) for 72 hours. For the final 24 hours of the treatment, doxycycline (1µg/mL) was added.


title: K562_DMSO_input_rep1


title: K562_GSK343_H3K9me3_rep2


title: bratmut_oocyte_rep_C

value: late stage oocyte


title: K562_GSK343_H3K9me3_rep1


title: K562_GSK343_H3K27me3_rep1


title: bratmut_oocyte_rep_A


title: bratmut_oocyte_rep_D


title: bratmut_oocyte_rep_B


title: K562_GSK343_H3K27me3_rep2


title: IKO2

city: Jinan

first: Yujia

last: Fan

address: Institute of Molecular Medicine and Genetics, School of Basic Medical Sciences, Shandong University, Jinan, Shandong, China.

institute: Shandong University

data_processing:
Raw data (raw reads) of fastq format were firstly processed through in-house perl scripts. In this step, clean data (clean reads) were obtained by removing reads containing adapter, reads containing ploy-N and low quality reads from raw data. At the same time, Q20, Q30 and GC content the clean data were calculated. All the downstream analyses were based on the clean data with high quality.
Reference genome and gene model annotation files were downloaded from genome website directly. Index of the reference genome was built using Hisat2 v2.0.5 and paired-end clean reads were aligned to the reference genome using Hisat2 v2.0.5. We selected Hisat2 as the mapping tool for that Hisat2 can generate a database of splice junctions based on the gene model annotation file and thus a better mapping result than other non-splice mapping tools.
featureCounts v1.5.0-p3 was used to count the reads numbers mapped to each gene. And then FPKM of each gene was calculated based on the length of the gene and reads count mapped to this gene. FPKM, expected number of Fragments Per Kilobase of transcript sequence per Millions base pairs sequenced, considers the effect of sequencing depth and gene length for the reads count at the same time, and is currently the most commonly used method for estimating gene expression levels.
Genome_build: HISAT2 v2.0.5
Supplementary_files_format_and_content: abundance measurements

extract_protocol:
Mouse intestine was cut longitudinally and washed three times with cold PBS. Villi were carefully scraped away and small pieces (5 mm) of intestine were incubated in 2 mM EDTA in PBS for 30 min on ice. These pieces were then vigorously suspended in cold PBS and the mixture was passed through 70 μm cell strainers.
RNA libaries were prepared for sequencing using standard Illumina protocols

treatment_protocol: Crypts were released from mice small intestine by incubation for 2mM EDTA in DPBS and leave on ice for 30 minutes. Crypts were re-suspended with cold Advanced DMEM/F12 media. Crypt cells were mixed with Matrigel and plated in 24-well plates. For passage, organoids were removed from Matrigel and mechanically dissociated into single-crypt domains, and then transferred to fresh Matrigel. Passage was performed every 1–2 weeks with a 1:5 split ratio.

tag: strain background

tag: tissue/cell type

value: organoid

tag: genotype/variation

value: KO


title: IWT2


title: IWT1


title: IKO1


title: K562_DMSO_H3K9me3_rep2

value: chronic myeloid leukemia


title: K562_DMSO_H3K27me3_rep2


title: B16N O1C44

value: Murine Melanoma Cells

value: Orai1 overexpressing

value: Orai1 overexpression

tag: time

value: Day 0


title: OKO2

treatment_protocol: Crypts were released from mice small intestine by incubation for 2mM EDTA in DPBS and leave on ice for 30 minutes. Crypts were re-suspended with cold Advanced DMEM/F12 media. Crypt cells were mixed with Matrigel and plated in 24-well plates. For passage, organoids were removed  from Matrigel and mechanically dissociated into single-crypt domains, and then transferred to fresh Matrigel. Passage was performed every 1–2 weeks with a 1:5 split ratio.

tag: background strain

value: Cullin 4b KO


title: K562_DMSO_H3K27me3_rep1

tag: cell type


title: K562_DMSO_H3K9me3_rep1


title: OWT2

tag: genotype

value: WT


title: B16N UV2

tag: cell line

value: WT, UV exposed

value: UV exposure

value: Week 2


title: B16N UV5

tag: treatment

value: Week 5


title: OKO1


title: RNASeq_KD_shHOTAIR2_rep3

city: Oslo

first: Philippe

last: Collas

address: PO Box 1112 Blindern

department: Institute of Basic Medical Sciences

country: Norway

institute: University of Oslo

data_processing:
RNA-Seq paired reads were aligned to the hg38 genome with Hisat2 (v2.1.0) using the ensembl annotation (v95).
Reads in genes were counted using featurecounts, with multimapping reads fractionally assigned using --fraction and -M.
Low abundance genes were filtered out of further analysis using edgeR's function "filterByExpr".
Normalisation between library sizes to get FPKM values was done using the trimmed mean of M-values method.
Normalisation for differential expression was by limma-voom (Law et al. 2014) and tests calculated by lmFit and eBayes.
Calculations of changing expression over time were done pairwise Prolif  v day0, day0 v day1, day1 v day3 and day3 v day9; first within each donor and then combined across donors.
Transcript quantification from RNA-Seq reads was conducted with salmon v1.3.0 (--numBootstraps 20 --gcBias) on an index of the ensembl transcriptome plus additional HOTAIR transcripts from refseq (NR_003716.3, NR_047517.1, NR_047518.1) and from the lncRNA sections of the ucsc browser (TCONS_00079053_HOTAIR, TCONS_00079054_HOTAIR,  TCONS_00079056_HOTAIR, TCONS_00079057_HOTAIR, TCONS_00079058_HOTAIR, TCONS_00079061_HOTAIR).
Genome_build: hg38 ensembl95
Supplementary_files_format_and_content: Bigwig RNA-Seq coverage file generated with deeptools bamCoverage (binsize 10bp)
Supplementary_files_format_and_content: Raw read counts per gene, one column per sample
Supplementary_files_format_and_content: FPKM for each gene passing the filtering threshold in each sample
Supplementary_files_format_and_content: TPM for each transcript in each gluteal sample

source_name: Human primary adipose stem cells

growth_protocol: Human normal adipose tissue-derived mesenchymal stem cells were isolated from subcutaneous liposuction material. Cells were cultured under proliferative conditions in DMEM/F12 containing 10% fetal calf serum, 10 ng/ml epidermal growth factor (Sigma-Aldrich E9644) and 20 ng/ml basic fibroblast growth factor (FGF2; Sigma-Aldrich F5392). Cells at passage 5-7 were used in three independent differentiations into the adipocyte lineage.

extract_protocol:
For RNA-seq, total mRNA was isolated from cells at aforementioned stages of differentiation using the Qiagen RNeasy kit and processed for Illumina library preparation and sequencing.
Illumina sequencing libraries were generated as per Illumina protocols.

treatment_protocol:
Two days before induction of differentiation, proliferating cells (day-2 [day minus 2] stage) were harvested and processed for RNA extraction. For adipogenic differentiation, harvested day-2 cells were reseeded in DMEM/F12/10% serum without growth factors. Adipogenesis was induced 48 h later (on day 0; D0) by addition of 10 µg/ml insulin, 200 µM indomethacin, 0.5 µM 1-methyl-3 isobutylxanthine and 1 µM dexamethasone. Medium was left with the cells for the 3 days of differentiation. Cells were collected on day0, day 1, day 3 and day9 (D0, D1, D3, D9) and processed for RNA extraction.
For transient knockdown, proliferating cells were electroporated with Nucleofector (Lonza) using the Human Mesenchymal Stem Cell NucleofectorTM Kit (Lonza). Two siRNAs (Silencer select from Ambion) were used - siHOTAIR 1: N272221, siHOTAIR 2: N272230. Electroporated cells were plated and cultured for 72 h in proliferative conditions before harvesting for RT-PCR and RNA-FISH.

value: Human primary adipose stem cells

tag: time point

tag: adipose depot

value: Gluteal

tag: culture

value: Cultured in vitro

tag: shRNA probe

value: shHOTAIR 2


title: RNASeq_KD_shHOTAIR1_rep3

value: shHOTAIR 1


title: RNASeq_SCR_Ctrl_rep3

value: scramble


title: LM47RN

type: RNA

city: Milan

first: Loris

last: De Cecco

address: via Venezian 1

country: Italy

institute: IRCSS Istituto Nazionale Tumori

hyb_protocol: Standard Affymetrix hybridization protocol

scan_protocol: Standard Affymetrix scanning protocol

data_processing: The data were processed and normalised using RMA with Transcriptome analysis Console software

label: biotin

source_name: human melanoma cell line

label_protocol: RNA from each sample was labeled according to the manufacturers recommendations (Thermo Fisher Scientific).

extract_protocol: RNA was extracted with QIAGEN RNeasy Lipid Tissue Mini kit in accordance with the prescribed protocol provided with the kit.

value: human melanoma cell line

tag: phenotype

value: Ra


title: LM20

value: Ri


title: B16N Control

data_processing:
Rsubread algorithm
Preprocessed data files from the samples were subject to RNA sequencing data analysis using Rsubread package
Assembly: GRCm38

value: Murine Melanoma Cells


title: IGR39


title: RPMI7951


title: SB


title: Single-cell RNA-seq dataset of human HEK293T cell line and NIH/3T3 cell line mixture, sublibrary 4

data_processing:
For single cell CH-RNA-seq. Sequenced reads were trimmed for adaptor sequence.Then reads in bam files were tagged with the cell and molecular (UMI) barcode sequences using SnapTools. The reads quality under 10 were removed. Cellbarcode:In read1, the bases 1-10,19-28 (NEO3,NEO4,META1,META2); the bases 1-10,42-51(NEO1,NEO2). UMI barcode:In read1, the bases 11-18(NEO3,NEO4,META1,META2); the bases 34-41(NEO1,NEO2).The raw fastq-format sequencing data from a DNBSEQ-T7 were first split into i7 indexed sub-libraries using splitBarcode (https://github.com/MGI-tech-bioinformatics/splitBarcode). For each sub-library, the revised Drop-seq core computational tool was used for data preprocessing  (http://mccarrolllab.org/wp-content/uploads/2016/03/Drop-seqAlignmentCookbookv1.2Jan2016.pdf). We used STAR (version 2.5.2a) with default parameters for mapping 3T3 cells and 293T cells. Reads from 3T3 cells and 293T cells were aligned to a merged hg19-mm10 reference genome (provided by Drop-seq group, GSE63269). To save mapping time , reads from axolotls were aligned to the axolotl genome (V3.0.0, https://www.axolotl-omics.org/assemblies) using another mapper bowtie. GTF annotation files were used to tag aligned reads. Cellular barcodes and unique molecular identifiers (UMIs) were directly extracted from Read1. A list of two rounds of cell barcode oligo sequences was used to correct the extracted cellular barcode from read1. Finally, the HTseq package was used to generate digital expression matrices of axolotl. For cell quality control of axolotl data, we excluded cells in which less than 200 transcripts were expressed. In the mixed-species experiment of 3T3 cells and 293T cells, Cellbarcode:In read1, the bases 1-10,42-51;UMI:In read1, the bases 34-41. The percentage of uniquely mapping reads for genomes of each species with over 85% UMIs assigned to one species was regarded as species-specific cells, while the remaining cells were labeled collisions. Count matrices of bulk RNA-seq of axolotl specimens were generated by featureCounts with default parameters.
For bulk RNA-seq, reads in bam file were aligned to the  genome assembly using STAR version (2.5.2a)  with default configurations. Next, merge the STAR alignment tagged bam were treated using DEseq2 with default configurationsand the reads are annotated with exon tags. Final featurecounts text files were used for downstream analysis.
Assembly: AmexG_v3.0.0(Axolotl genome assembly v3.0.0)(https://genome.axolotl-omics.org/cgi-bin/hgTracks?db=ambMex3&lastVirtModeType=default&lastVirtModeExtraState=&virtModeType=default&virtMode=0&nonVirtPosition=&position=AMEXG_0030000001%3A1000%2D2000&hgsid=43238_8SJNnizHOy6T42X4YaUpQyigV8RT)or(https://www.axolotl-omics.org/assemblies)
Supplementary files format and content: tab-delimited text files of digital expression matrix (dge)(single-cell RNA-seq)

organism:
Homo sapiens
Mus musculus

extract_protocol:
Total RNA of each tissue was purified using standard TRIzol based protocols for bulk RNA-seq;For single-cell RNA-seq, frozen cells of each tissue were thawed at 37 °C and centrifuged at 500×g for 5 min first. Then, fresh fixed or thawed frozen cells were resuspended with 100 µl PBS, and 400 µl Triton X-100 in PBS (10 µl 10% Triton X-100 in 390 µl PBS) was added. The mixture was incubated on ice for 3 min for permeabilization. Next, the cells were pelleted and resuspended with 500 µl DEPC-treated water, and 3 ml 0.1 N HCl was added slowly. After 5 min of incubation on ice, 3.5 ml Triton X-100 in Tris-HCl (35 µl 10% Triton X-100 in 3.5 ml 1 M Tris-HCl, pH 8.0) was added to quench permeabilization. Cells were then washed once with 1 ml of PBS and filtered with a 40 μm strainer. Cell density was determined using a blood cell counting chamber.
For bulk RNA-seq, RNA extraction followed reverse transcription, second strand DNA synthesis, transposase tagmentation and standard Illumina Nextera library construction. For single-cell RNA-seq, cell numbers for each tissue were estimated, and the corresponding volume of cells in PBS was split into four 96-well plates. For each well, 6.5 µl cells (from 5000 to 20000) was mixed with 0.5 µl 10 mM dNTPs (Thermo Fisher Scientific) and 0.5 µl 100 μM RT barcode primer (see Table S1). The mixture was incubated at 65 °C for 5 min and immediately placed on ice. Two microliters of 5X RT buffer (31 mM Tris-HCl, pH 8.0, 37.5 mM NaCl, 3.1 mM MgCl2, 10 mM DTT), 0.5 µl Maxima H Minus RTase (Thermo Fisher Scientific) and 0.1 µl Murine RNase inhibitor were premixed, and 2.5 µl RT mixture was added to each well. Reverse transcription was carried out by incubating the plates at gradient temperature: 3 cycles (8℃ for 12 s, 15℃ for 45 s, 20℃ for 45 s, 30℃ for 30 s, 42℃ for 2 min, and 55℃ for 3 min) and 55℃ for 60 min. After reverse transcription, plates were placed on ice for 1 min to stop the reaction. All reagents were pooled together into a 15 ml centrifuge tube and then centrifuged at 500 g for 5 min. Pellet was washed with PBS twice and resuspended with hybridization buffer (50 mM Tris-HCl, 10 mM MgCl2, 10 mM DTT, 0.1% Triton X-100, 10% PEG8000 (Sigma-Aldrich), 1% Murine RNase inhibitor). Hybridization buffer within cells was split into eight 96-well plates (3 µl for each well) and 2 µl 25 μM pre-annealing hybridization primers (50 μM HY head oligo, 50μM barcoded HY primer oligo, mixed equally and incubated at 95℃ for 2 min, then slowly cooled to 25℃ with a temperature ramp of −0.1℃/s) were added to each well (see in Table S1). Plates were incubated at 37 °C for 90 min, and 0.5 µl of 100 μM block tail primer oligo was added to block any redundant hybridization primers. After blocking for 30 min at 37 °C, all reagents were pooled into a 15 ml centrifuge tube and then centrifuged at 500 g for 5 min. The pellet was washed with PBS twice and resuspended in 40 µl of PBS. Then, 60 µl PNK mix (10 µl 10X PNK buffer (NEB), 20 µl T4 polynucleotide kinase (NEB), 10 µl 10 mM ATP (NEB), and 20 µl DEPC-treated water) was added. The PNK reaction was carried out at 37℃ for 30 min. Then, 1 ml ice-cold PBS was added to stop the reaction. Cells were then filtered using a 40μm strainer. The reagent was centrifuged at 500 g for 5 min after discarding the supernatants. The pellet was again resuspended in PBS, and the density of cells was estimated. After splitting into one or more 96-well plates. There were 5000~8000 cells per well. The volume was adjusted to 8 µl. Second strand synthesis mix (1.33 µl 10X buffer, 0.66 µl second strand synthesis enzyme mix) was added into each well and the plates were incubated at 16℃ for 3 hours (stop point at 4℃). Then, 10 µl cell lysis buffer (20 mM Tris pH 8.0, 400 mM NaCl, 100 mM EDTA, 4.4% SDS (Sangon Biotech)) and 2 µl proteinase K (Sangon Biotech) were added to the wells. Cell lysis was performed at 55℃ for 60 min. Then, 1 µl PMSF (1 mM) was added to quench the lysis reaction. The plates were incubated at 37℃ for 10 min. For each well, the mixture was then purified using 1.5X VAHTS DNA Clean Beads (Vazyme Biotech). Then, 10 µl of product in DEPC-treated water was transferred into new 96-well plates. For each plate, we randomly chose 10 wells to quantify the dsDNA concentration using Equalbit3.0. Tn5 transposase from the TruePrep DNA Library Prep Kit V2 (Vazyme Biotech) was used for cDNA tagmentation. Then, 10 µl of product in DEPC-treated water was transferred into a new 96-well plate. For each well, 14 µl PCR mix (12 µl 2X KAPA HiFi HotStart ReadyMix (Kapa Biosystems), 1 µl 10 mM P5 primer, and 1 µl 10 mM indexed P7 primer, see Table S1) was added to the plates. The PCR program was as follows: 72 °C for 5 min; 98 °C for 30 s; 12 cycles of 98 °C for 10 s, 60 °C for 30 s, and 72 °C for 1 min; 72 °C for 5 min; and a 4 °C hold. After pooling PCR products, two rounds of size selection with VAHTS DNA clean beads (Vazyme Biotech) were used to purify the DNA library between 300 and 500 bp. The libraries were finally mixed, and the concentration of dsDNA was quantified.The purified linear DNA library was circularized into single strand DNA (ssDNA) library using VAHTS® Circularization Kit for MGI (Vazyme). Then ssDNA library was amplified using DNBSEQ DNB preparation kit (MGI). Amplified DNA nanoball (DNB) was sequenced with custom primers on MGI DNBSQ-T7 with dark reaction model ( Single-cell RNA-seq: 100 cycles of read1 with dark reaction from 11-33, 100 cycles of read2;For NEO1,NEO2: PE150, no dark  reaction). Human and mouse cell line libraries were sequenced with custom primers on Illumina Hiseq Xten with pair-end 150bp mode.

tag: strain

tag: age

tag: tissue

value: HEK293T and NIH/3T3


title: LM38


title: M308


title: M233


title: LM11R


title: LM42N

value: S


title: KO4

source_name: small intestine

value: crypt

value: Cullin 4b KO


title: RNASeq_KD_shHOTAIR2_rep2

tag: chip antibody


title: Single-cell RNA-seq dataset of human HEK293T cell line and NIH/3T3 cell line mixture, sublibrary 3


title: LM47R


title: LM11


title: OWT1

value: organoid

value: WT


title: LM47


title: KO3


title: RNASeq_KD_shHOTAIR1_rep2


title: Single-cell RNA-seq dataset of human HEK293T cell line and NIH/3T3 cell line mixture, sublibrary 2


title: KO1


title: Single-cell RNA-seq dataset of human HEK293T cell line and NIH/3T3 cell line mixture, sublibrary 1


title: ISL1CKO_P9_6

data_processing:
Raw reads were trimmed of Illumina sequencing adaptor and bases with reading quality lower than 20 using cutadapt v1.18 49. Reads shorter than 20 bp were filtered out.
Ribosomal RNA and reads mapping to UniVec database were filtered out using bowtie v1.2.2. with parameters -S -n 1 and SortMeRNA database
FASTQ files were mapped to the mouse genome using STAR [version 2.7.0c 48] GRCm38 primary assembly and annotation version M8
Count table was generated by Rsubread v2.0.1 package using default parameters without counting multi-mapping reads
Assembly: GRCm38.p6 (Ensembl)
Supplementary files format and content: Count_matrix_E14.5.txt.gz: Compressed tab seprated text file containing raw counts for control and Isl1 knockout samples of E14.5. Rows corresponds individual genes with ensembl ID and columns to samples.
Supplementary files format and content: Count_matrix_P9.txt.gz: Compressed tab separated text file containing raw counts for control and Isl1 knockout samples of P9. Rows corresponds individual genes with ensembl ID and columns to samples.

source_name: Pancreas

extract_protocol:
The pancreata microdissected from E14.5 embryos were directly trypsinized and prepared for FACS. Briefly, tissue was dissociated using 0.05% trypsin/0.53 mM EDTA at 37oC for 5 min. Digestion was stop by the FACS buffer (2% FBS and 10 mM EGTA in PBS 46), and cells were then 1x washed by FACS buffer. The pancreata from P9-P10 pups were first perfused by 1mg/ml collagenase in Hank’s balanced salt solution, dissected, and incubated at 37oC for 10-12 min to release endocrine cells/islets. Digested pancreatic tissue was washed 3x by 1% FBS in Hank’s solution. To generate single cells, the tissue was further dissociated by trypsinization as described above. Finally, cell suspensions were filtered through 40 μm nylon mesh and immediately tdTomato+ cells were sorted using a flow cytometer (BD FACSAriaTM Fusion), through a 100 μm nozzle in 20 psi, operated with BD FACSDiva™ Software.
For RNA sequencing, 100 sorted cells were collected into individual wells of 96-well plate containing 5 μl of lysis buffer of NEB Next single-cell low input RNA library prep kit for Illumina (New England Biolabs #E6420).

value: Pancreas

value: endocrine cells

value: Isl1 knockout


title: LM17R


title: KO2


title: RNASeq_KD_shHOTAIR1_rep1

value: Human primary adipose stem cells

value: Gluteal

value: Cultured in vitro


title: RNASeq_KD_shHOTAIR2_rep1

value: shHOTAIR 2


title: WT4


title: WT3


title: ISL1CKO_P9_5


title: WT2


title: RNASeq_SCR_Ctrl_rep2

value: scramble


title: ISL1CKO_P9_3


title: RNASeq_SCR_Ctrl_rep1


title: WT1


title: LM43R

value: human melanoma cell line

value: Ra


title: K562_GSK343_RNAseq_rep3

data_processing:
Adapter contamination, polyA read through, and low quality tails were removed using bbduk (version 38.23) with the following parameters: ref=polyA.fa.gz,truseq_rna.fa.gz k=13 int=f ktrim=r useshortkmers=t mink=5 qtrim=r trimq=10 minlength=20.
Read mapping was performed with STAR (version 2.6.0c)  with the following parameters: --genomeDir Homo_sapiens.GRCh38.dna.primary_assembly.fa --outFilterType BySJout --outFilterMultimapNmax 20 --alignSJoverhangMin 8 --alignSJDBoverhangMin 1 --outFilterMismatchNmax 999 --outFilterMismatchNoverLmax 0.1 --alignIntronMin 20 --alignIntronMax 1000000 --alignMatesGapMax 1000000 --outSAMattributes NH HI NM MD.
Reads aligned over annotated genes were quantified using HTSeq (version 0.11.2) using the following command: htseq = count -s yes.
Changes in expression calculated through DEseq2 are detailed in the tab-delimited text file: K562_EZH2_DMSO_Differential.txt, containing Transcript_ID in the first column and values for expression change as well as statistical significance in the subsequent columns.
Genome_build: hg38
Supplementary_files_format_and_content: The HTSeq raw count files for each individual sample are provided as well as a summary table, "K562_EZH2_DMSO_Differential.csv", describing the expression changes upon EZH2 inhibition.

source_name: Flag-NLS-M.CviPI-Lamin-B1 K562 clonal cells, EZH2i 72 hours, Doxycycline 24 hours

extract_protocol:
RNA was harvested using the RNeasy Plus Mini Kit (Qiagen).
RNA libraries were prepared using the QuantSeq 3' mRNA-Seq Library Prep Kit FWD for Illumina (Lexogen).

treatment_protocol: Cells were seeded at 125,000 cell/mL and then treated with DMSO (0.1% v/v) or GSK343 (1µM) for 72 hours. For the final 24 hours of the drug treatment, doxycycline (1µg/mL) was added. Cells were then centrifigued and washed twice with cold PBS before being snap frozen.

value: chronic myeloid leukemia

value: GSK343 (1µM) for 72 hours and induced with doxcycline (1µg/mL) for the final 24 hours of treatment


title: ISL1CKO_P9_4


title: ISL1CKO_P9_2


title: LM36

value: S


title: LM36R


title: K562_GSK343_RNAseq_rep2

value: Flag-NLS-M.CviPI-Lamin-B1 K562 clonal cells


title: LM43


title: ISL1CKO_P9_1


title: K562_GSK343_RNAseq_rep1