title: 3543_OF1A_7-5-C10-F10


title: 3543_OF1A_7-5-C10-F9

status: Public on Dec 13 2023

type: SRA

city: La Jolla

first: Joseph

middle: R

last: Ecker

state: CA

address: 10010 North Torrey Pines Road

department: Genomic Analysis Laboratory

country: USA

institute: HHMI-Salk-Institute

library_source: genomic

data_processing:
Reads trimmed with Cutadapt for adapters and low quality bases (Phred score >= 20)
The resulting files are mapped to in-silico bisulfite converted hg38 reference genome using Hisat-3n.
The raw BAM files were filtered using samtools to keep only uniquely mapped reads and use Picard to remove PCR duplicates.
From the filtered BAM file, DNA reads are selected by read-level mCH base coverage >= 3 and read-level mCH fraction < 0.5, selected reads will be processed by ALLCools package to produce ALLC files (allcools allc-to-bam function). The ALLC files are Tab-delimited files describing the quantity of unmethylated and methylated cytosine base calls.
Trimmed reads will also be mapped by Hisat2 to map to hg38 reference genome with GENCODE human v37 gene annotation. RNA reads are selected by read-level mCH base coverage >= 3 and read-level mCH fraction > 0.9, selected reads will be processed by featureCount to quantify gene level (exon + intron) RNA counts.
Assembly: hg38
Supplementary files format and content: tab-delimited files (ALLC files) describing the quantity of unmethylated and methylated cytosine base calls. Columns are: 1. Chromosome, 2. Position, 3. Strand, 4. Cytosin Context, 5. methylated base call, 6. total mapped base call, 7. not used, place holder column.
Library strategy: snmCT-seq

molecule: genomic DNA

organism: Homo sapiens

source_name: OF1A human BA46

extract_protocol: Library prepared following protocol discribed in Luo, Chongyuan, Hanqing Liu, Fangming Xie, Ethan J. Armand, Kimberly Siletti, Trygve E. Bakken, Rongxin Fang, et al. 2019. “Single Nucleus Multi-Omics Links Human Cortical Cell Regulatory Genome Diversity to Disease Risk Variants.” bioRxiv. https://doi.org/10.1101/2019.12.11.873398. https://www.protocols.io/view/snmcat-v1-bwubpesn

tag: brain region

value: Dorsolateral Prefrontal cortex (Brodmann area 46)

tag: age

tag: Sex

value: Female

tag: molecule subtype

value: nuclear RNA + genomic DNA


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title: 3543_OF1A_6-5-I2-I21


title: 3543_OF1A_6-5-I2-I10


title: 3543_OF1A_6-5-I2-H22


title: 3543_OF1A_6-5-I2-H21


title: 3543_OF1A_6-5-I2-H10


title: 3543_OF1A_6-5-I2-G9


title: 3543_OF1A_6-5-I2-G22


title: 3543_OF1A_6-5-I2-G10


title: 3543_OF1A_6-5-I2-F9


title: 3543_OF1A_6-5-I2-G21


title: 3543_OF1A_6-5-I2-F22


title: 3543_OF1A_6-5-I2-D9


title: 3543_OF1A_6-5-I2-F21


title: 3543_OF1A_6-5-I2-E10


title: 3543_OF1A_6-5-I2-D22


title: 3543_OF1A_6-5-I2-E22


title: 3543_OF1A_6-5-I2-D21


title: 3543_OF1A_6-5-I2-C9


title: 3543_OF1A_6-5-I2-D10


title: 3543_OF1A_6-5-I2-B9


title: 3543_OF1A_6-5-I2-C22


title: 3543_OF1A_6-5-I2-B10


title: 3543_OF1A_6-5-I2-C10


title: 3543_OF1A_6-5-I2-H9


title: 3543_OF1A_6-5-I2-B22


title: 3543_OF1A_6-5-I2-A22


title: 3543_OF1A_6-5-I2-B21


title: 3543_OF1A_6-5-I2-C21


title: 3543_OF1A_6-5-I2-A21


title: 3543_OF1A_6-5-I2-A10


title: 3543_OF1A_6-4-I2-P7


title: 3543_OF1A_6-5-I2-E9


title: 3543_OF1A_6-5-I2-F10


title: 3543_OF1A_6-4-I2-P20


title: 3543_OF1A_6-5-I2-E21


title: DP-MSC2 with DDR2 signal

city: Taichung

first: Wei-Chung

last: Cheng

address: No. 91, Hsueh-Shih Road

department: Research Center for Tumor Medical Science

country: Taiwan

institute: China Medical University

library_source: transcriptomic

data_processing:
1. trim adapter: trimmomatic
2. Alignment: Hisat2
3. The quantification were performed by featureCounts 2.0.0.
4. Expression profiling: stringtie
hisat2_index: V97
Assembly: Homo_sapiens.GRCh38
Supplementary files format and content: tab-delimited text file includes TPM values for each Sample

molecule: total RNA

source_name: Lower anterior deciduous teeth

growth_protocol: Mononuclear cells were isolated by density gradient centrifugation using Ficoll-Hypaque (Sigma-Aldrich) at a density of 1.077 g/L, followed by culturing in Dulbecco’s Modiﬁed Eagle Medium (DMEM; Gibco-BRL) supplemented with 16.6% fetal bovine serum (FBS, Gibco-BRL), 100 units/mL penicillin, 100 µg/mL streptomycin, and 2 mM L-glutamine (Invitrogen) at 37◦C under 5% CO2 atmosphere.

extract_protocol:
Total RNA of bone marrow (BM-hMSCs) and dental pulp (DP-hMSCs) with 40 uM BiFP containing integrin α2β1 (GFOGER) or discoidin domain receptors 2 (DDR2, GVMGFO) binding motifs was isolated with the Qiagen RNeasy Kit (QIAGEN).
VAHTS Universal V6 RNA-seq Library Prep kit was used to prepare RNA-Seq library.

treatment_protocol: Three individual hMSCs from distinct sources, such as bone marrow (BM-hMSCs) and dental pulp (DP-hMSCs), were cultured in complete growth medium with 40 uM BiFP containing integrin α2β1 (GFOGER) or discoidin domain receptors 2 (DDR2, GVMGFO) binding motifs for 48 h.

tag: tissue

value: Lower anterior deciduous teeth

tag: cell line

value: Primary Cells

tag: cell type

value: Mesenchymal stem cells

tag: genotype

value: WT

tag: treatment

value: 40 uM BiFP containing discoidin domain receptors 2 (DDR2, GVMGFO)


title: DP-MSC3 with DDR2 signal


title: DP-MSC3 with Control signal

value: 40 uM Control peptide


title: DP-MSC3 with Integrin signal

value: 40 uM BiFP containing integrin α2β1 (GFOGER)


title: DP-MSC2 with Integrin signal


title: DP-MSC2 with Control signal


title: BM-MSC3 with DDR2 signal

source_name: Bone marrow

value: Bone marrow


title: DP-MSC1 with Control signal


title: DP-MSC1 with DDR2 signal


title: BM-MSC3 with Control signal


title: DP-MSC1 with Integrin signal


title: 3543_OF1A_6-5-I2-A9


title: BM-MSC3 with Integrin signal


title: BM-MSC2 with DDR2 signal


title: BM-MSC2 with Control signal


title: BM-MSC2 with Integrin signal


title: BM-MSC1 with DDR2 signal


title: BM-MSC1 with Control signal


title: BM-MSC1 with Integrin signal


title: 3543_OF1A_6-4-I2-P8


title: NCI-H1299/CDDP Input,resistant, H3K27me3, rep1

city: Shenyang

first: Qiuyue

last: Zhang

address: No. 103, Wenhua Road, Shenhe District

country: China

institute: Shenyang Pharmaceutical University

data_processing:
Raw sequencing data was first filtered by fastp (version 0.23.4), low-quality reads were discarded and the reads contaminated with adaptor sequences were trimmed.
The clean reads were used for protein binding site analysis. They were mapped to the reference genome of GRCh38 using STAR software (version 2.5.3a) with default parameters.
The MACS2 software（Version 2.1.1）was used for peak calling. The bedtools（Version 2.25.0）was used for peaks annotation and peak distribution analysis.
The differentially binding peaks were identified by csaw(version 1.30.1).
The Homer（version 4.10）was used for motifs analysis.
Assembly: GRCh38
Supplementary files format and content: Bigwig

source_name: lung

extract_protocol:
The cell was fixed in 1% formaldehyde for 10 min at room temperature. After quenching cell was treated with cell lysis buffer and nucleus was collected by centrifuging at 2000g for 5min. Then, nucleus was treated with nucleus lysis buffer and sonicated to fragment chromatin DNA.
The high-throughput DNA sequencing libraries were prepared by using VAHTS Universal DNA Library Prep Kit for Illumina V3（Catalog NO. ND607, Vazyme）. The library products corresponding to 200-500 bp were enriched and quantified.

value: lung

value: Non-small cell lung cancer cell line

tag: antibody


title: NCI-H1299/CDDP IP,resistant, H3K9me2, rep1


title: NCI-H1299/CDDP IP,resistant, H3K27me3, rep1


title: NCI-H1299 Input, parental, H3K27me3, rep1


title: NCI-H1299 Input, parental, H3K9me2, rep1


title: NCI-H1299/CDDP Input,resistant, H3K9me2, rep1


title: NCI-H1299 IP, parental, H3K27me3, rep1


title: NCI-H1299 IP, parental, H3K9me2, rep1


title: Y24F2

city: Lausanne

first: Paolo

last: Angelino

address: Quartier Sorge, Bâtiment Génopode

department: BCF

country: Switzerland

institute: SIB Swiss Institute of Bioinformatics

data_processing:
raw data have been processed with the workflow: nf-core/rnaseq, 3.12.0
read trimming: trimgalore, 0.6.7
gene expression quantification: salmon, 1.10.1
Assembly: Homo_sapiens, GRCh38, ENSEMBL V. 98
Supplementary files format and content: tab separated values, matrix of raw counts

source_name: Blood

growth_protocol: Primary CD4+ T cells were cultured at a concentration of 106 cells/mL in RPMI-1640 (Thermo Fisher Scientific) supplemented with 10% HI-FBS and 50 µg/mL gentamicin at 37°C, 5% CO2.

extract_protocol:
Total RNA was extracted from 500,000 cells using Quick-RNA Miniprep Kit (Zymo Research) according to manufacturer’s recommendations.
RNA libraries were prepared using Illumina stranded mRNA protocol

treatment_protocol: CD4+ T cells were stimulated by adding 25 µL/mL ImmunoCult Human CD3/CD28 T Cell Activator (Stemcell Technologies) in medium supplemented with IL-2 [200 IU/mL]

value: Blood

value: CD4+ T Cells

value: F

tag: donor

value: Y

tag: time

tag: batch


title: X24M1

value: M

value: X


title: X72M1


title: W24F2

value: W


title: W24F1


title: W72F1


title: V72M1

value: V


title: V24M1


title: W72F2


title: U72F1

value: U


title: U24F1


title: R72M2

value: R


title: P72M2

value: P


title: Q72F2

value: Q


title: O72F2

value: O


title: P24M2


title: O24F2


title: Y72F2


title: M24F1


title: M72F1


title: H24M2

value: H


title: S24F2

value: S


title: H72M2


title: E72F1

value: E


title: F72M1


title: F24M1


title: Delta24M2

value: Delta


title: E24F1

value: Blood


title: B72M1

value: CD4+ T Cells

value: B


title: Beta72M1

value: Beta


title: Delta72M2

value: M


title: B24M2


title: D72M1

value: D


title: S72F2

value: F


title: Q24F2


title: R24M2


title: D24M1


title: Beta24M1


title: B24M1


title: Alpha24F2

value: Alpha


title: Alpha72F2


title: A72F1

value: A


title: Donor S, male

library_source: transcriptomic single cell

data_processing:
The Cell Ranger Single Cell Software Suite 7.0.0 was used to perform sample demultiplexing, barcode processing, and single cell 5’ gene counting for transcriptome with refdata-gex-GRCh38-2020-A as reference
Feature list used to run CellRanger on TotalSeqC assay:  CD4,CD4_TotalSeqC,R2,5PNNNNNNNNNN(BC)NNNNNNNNN,GAGGTTAGTGATGGA,Antibody Capture CD69,CD69_TotalSeqC,R2,5PNNNNNNNNNN(BC)NNNNNNNNN,GTCTCTTGGCTTAAA,Antibody Capture
Assembly: GRCh38
Supplementary files format and content: Tab-separated values files and matrix files

extract_protocol:
Library was performed according to the manufacturer's recommendations (single cell 5' v2, 10X Genomics). Briefly, cells were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was retrotranscripted to cDNA, which contains an Illumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10X barcode. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the apropriate sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Illumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index was added
scRNA-seq + TotalSeqC


title: Donor M, male


title: Donor Delta, male


title: Donor Beta, male


title: B72M2


title: Monocytes, COVID-19 EN70

city: Muenchen

first: Bing

last: Han

address: Marchioninistrasse 15

country: Germany

description: Monocytes, COVID-19

data_processing:
Sequence reads were trimmed and checked for Quality using fastp v0.23.1
Trimmed sequence reads were mapped to to GRCh38 with HISAT2 v2.1.2
Transcripts were quantified by Rsubread v1.22.2
Assembly: GRCh38
Supplementary files format and content: Comma-separated values include RPKM values for each Sample

source_name: Separated Monocytes

extract_protocol:
Monocytes were isolated using StraightFrom® Whole Blood CD14 MicroBeads from Miltenyi Biotec  for separation. Total RNA was extracted from separated monocytes using NucleoSpin RNA/Protein kit
RNA libraries for RNA-seq were prepared by Eurofins Genomics.

value: Separated Monocytes


title: Monocytes, COVID-19 EN83

description: Monocytes, COVID-19


title: Monocytes, COVID-19 EN69

description: Monocytes, COVID-19


title: Monocytes, CONTROL EN98

description: Monocytes, CONTROL

value: CONTROL


title: Monocytes, CONTROL EN99

description: Monocytes, CONTROL


title: Monocytes, COVID-19 EN96

description: Monocytes, COVID-19


title: Monocytes, CONTROL EN78

description: Monocytes, CONTROL


title: HEK293T, WT, input

city: Farmington

first: Duomeng

last: Yang

address: 263 Farmington Avenue

institute: Uconn health

data_processing:
ChIP-seq reads were aligned to human reference assembly GRCh38 using Bowtie2 v2.3.5.1 aligner
Peaks were identified by comparing the ChIP and input data using MACS2 v2.1.2
Assembly: hg38
Supplementary files format and content: narrowPeak file includes peak calling result for each sample

extract_protocol:
The cells were cross-linked with 1% formaldehyde for 10 min at 37°C, and then we used a ChIP-IT® Express Enzymatic Shearing Kit (ActiveMotif, Cat #53035) combining with a ChIP assay kit (Millipore-Sigma, Cat #17-295) and a ChIP DNA Clean & Concentrator kit (ZYMO research, Cat #D5205) to perform the ChIP experiments following the manufactor's protocol.
ChIP-seq libraries were prepared by Yale Center for Genome Analysis

value: Human embryonic kidney cells

value: WT

tag: chip antibody

value: VSV


title: HEK293T, UBR5KO-VSV-1,ChIP

value: TRIM28 (BioLegend, #619302)


title: HEK293T, WT-VSV-1,ChIP


title: H9_XIST_KO_C18_H3K27me3_R2

city: Los Angeles

first: Kathrin

last: Plath

institute: UCLA

description: CUT&Tag using H3K27me3 antibody in H9 with XIST KO (clone 18) (Rep2)

data_processing:
Trim: trim_galore (v0.4.1) with default parameters was used to trim DNA sequencing reads
Align: Bowtie2 (v2.2.9) with the default parameters was used to align reads
Filter: Samtools (v1.9) was used to remove reads with mapping quality less than 30, or marked by Picard MarkDuplicates (v2.1.0) as PCR duplicates
Count: Bedtools (v2.26.0) intersect was used to count reads in each genomic region (100kb windows every 25kb)
Enrichment: read counts in each window were normalized by the total read counts in each sample. The ratio of the normalized reads in the pulldown to the input were used as inputs
Assembly: hg38
Supplementary files format and content: bedgraph files represent normalized enrichment ratios in windows across the genome (100kb windows every 25kb). Is_unmappable=yes mark unmappable genomic regions
Library strategy: CUT&Tag

growth_protocol: Female (H9 WT and two clones of XIST KO) and male (WIN1) naïve hESCs  were cultured in 5iLAF naïve ESC medium on irradiated male mouse embryonic fibroblasts (1:1 mixture of DMEM/F12 and Neurobasal supplemented with N2, B27, penicillin/streptomycin, nonessential amino acids, GlutaMAX, 0.5% KSR, 0.1mM β-mercaptoethanol, 50ug/ml bovine serum albumin, 20ng/ml rhLIF, 20ng/ml Activin A, 8ng/ml FGF2, 1uM MEK inhibitor PD0325901 0.5uM B-Raf inhibitor SB590885, 1uM GSK3-β inhibitor IM-12, 1uM Src inhibitor WH-4-023, and 10uM ROCK inhibitor Y-27632). hESCs were cultured in 5% CO2, 5% O2 (Hypoxia) at 37C and

extract_protocol:
Samples are prepared by following CUT&Tag protocol.
Library construction was conducted by following Hatice Kaya-Okur et.al. 2019 Nature Communications, briefly, extracted genomic DNA was PCR amplified by using NEB Nextera primers and NEB Next HiFi PCR master mix for 13 cycles including a 10 seconds combined anealing/extension step.

treatment_protocol: In addition to growing each cell type in appropriate media condition, cells were not treated with any additional agent(s).

value: Naive hESC

tag: library strategy

value: CUT&Tag

tag: experiment type

value: H3K27me antibody


title: H9_XIST_KO_C18_H3K27me3_R1

description: CUT&Tag using H3K27me3 antibody in H9 with XIST KO (clone 18) (Rep1)


title: HEK293T, UBR5KO, input


title: HEK293T, WT-VSV-2,ChIP


title: A24F1

value: Blood

value: CD4+ T Cells


title: H9_XIST_KO_C18_IgG_R1

description: CUT&Tag using IgG control in H9 with XIST KO (clone 18) (Rep1)

value: IgG


title: H9_XIST_KO_C7_H3K27me3_R2

description: CUT&Tag using H3K27me3 antibody in H9 with XIST KO (clone 7) (Rep2)


title: H9_XIST_KO_C7_H3K27me3_R1

description: CUT&Tag using H3K27me3 antibody in H9 with XIST KO (clone 7) (Rep1)


title: H9_XIST_KO_C7_IgG_R2

description: CUT&Tag using IgG control in H9 with XIST KO (clone 7) (Rep2)


title: ALL cells, NRIP1-PDGFRB

city: Tokyo

first: Yosuke

last: Tanaka

address: 5-1-1, tsukiji, Chuo-ku

country: Japan

institute: National Cancer Center Research Institute

data_processing:
Illumina Casava1.8.2 software used for basecalling.
Paired-end reads were aligned to the hg38 human genome and were quantified using STAR (https://github.com/infphilo/tophat) and RSEM (https://github.com/deweylab/RSEM)
Assembly: hg38
Supplementary files format and content: tab-delimited text file includes FPKM values for the Sample.

source_name: Human leukemic cells

extract_protocol:
RNA with a high RNA integrity number underwent RNA-seq using a NEBNext Ultra Directional RNA Library Prep Kit.
RNA libraries were prepared for sequencing using standard Illumina protocols

value: Human leukemic cells

tag: disease state

value: acute lymphoblastic leukemia (ALL)


title: H9_XIST_KO_C18_IgG_R2

description: CUT&Tag using IgG control in H9 with XIST KO (clone 18) (Rep2)


title: H9_XIST_KO_C7_IgG_R1

description: CUT&Tag using IgG control in H9 with XIST KO (clone 7) (Rep1)


title: H9_WT_H3K27me3_R1

description: CUT&Tag using H3K27me3 antibody in WT H9 (Rep1)


title: H9_WT_IgG_R1

description: CUT&Tag using IgG control in WT H9 (Rep1)


title: WIN1_H3K27me3_R2

description: CUT&Tag using H3K27me3 antibody in WIN1 (Rep2)


title: H9_WT_IgG_R2

description: CUT&Tag using IgG control in WT H9 (Rep2)


title: Monocytes, CONTROL EN100

description: Monocytes, CONTROL


title: WIN1_H3K27me3_R1

description: CUT&Tag using H3K27me3 antibody in WIN1 (Rep1)


title: HEK293T, UBR5KO-VSV-2,ChIP


title: WIN1_IgG_R2

description: CUT&Tag using IgG control in WIN1 (Rep2)


title: WIN1_IgG_R1

description: CUT&Tag using IgG control in WIN1 (Rep1)


title: Human male naive hESC WIN12 - INPUT

description: ChIP-seq input (used as control) in female navie hESCs (WIN1)

data_processing:
Align: Bowtie2 (v2.2.9) with the default parameters was used to align reads
Filter: Reads that aligned to a unique position with no more than two sequence mismatches were retained. Multiple reads mapping to the exact same location and strand in the genome were collapsed to a single read to account for clonal amplification effects.
Count: Bedtools (v2.26.0) intersect was used to count reads in each genomic region (100kb windows every 25kb)
Enrichment: read counts in each window were normalized by the total read counts in each sample. The ratio of the normalized reads in the pulldown to the input were used as inputs
Assembly: hg38
Supplementary files format and content: bedgraph files represent normalized enrichment ratios in windows across the genome (100kb windows every 25kb). Is_unmappable=yes mark unmappable genomic regions
Supplementary files format and content: bedgraph files with chromatin states

growth_protocol: Female naïve iPSCs and WIN1 male naïve hESCs used in ChIP-seq experiment were cultured in 5iLAF media in conditions previously described (Theunissen et al., 2014). 5iLAF media consisted of a 1:1 mixture of DMEM/F12 and Neurobasal (Life Technologies), supplemented with 1x N2 (Life Technologies), 1x B27 (Life Technologies), 1x penicillin/streptomycin, 1x nonessential amino acids, 0.5x GlutaMAX, 0.5% KSR, 0.1mM βmercaptoethanol, 50µg/ml bovine serum albumin (Sigma), 20ng/ml rhLIF (EMD Millipore), 20ng/ml Activin A (Peprotech), 8ng/ml FGF2, 1µM MEK inhibitor PD0325901 (Stemgent or Bio-Techne), 0.5µM B-Raf inhibitor SB590885 (Bio-Techne), 1µM GSK3β inhibitor IM-12 (Enzo), 1µM Src inhibitor WH-4- 023 (A Chemtek), and 10µM ROCK inhibitor Y-27632. For passaging, cells were dissociated by a 3 min treatment with StemPro Accutase (Life Technologies) at 37°C and re-plated after passing through a 40µm cell strainer in 5iLAF medium. Naïve hESCs were passaged as single cells every 5−6 days. In general naïve hPSCs were grown on irradiated DR4 mouse embryonic fibroblast feeder cells and maintained in a humidified 37°C incubator at 5% CO2 and atmospheric oxygen levels. Prior to ChIP experiments, a feeder depletion step was introduced by allowing feeder cells to attach to plastic surfaces for 40 min at 37oC and collecting only the non-attached PSC fraction.

extract_protocol:
Histone Modifications were assesed by means of X-linked ChIP
Histone modification genome-wide position data generated in this study were acquired using ChIP after crosslinking cells.  Briefly, cells were grown to a final concentration of 5x107 cells for each ChIP-seq experiment. Cells were chemically cross-linked at room temperature by the addition of formaldehyde to 1% final concentration for 10 minutes and quenched with 0.125 M final concentration glycine. Cross-linked cells were re-suspended in sonication buffer (50mM Hepes-KOH pH 7.5, 140mM NaCl, 1mM EDTA, 1% TritonX-100, 0.1% Na-deoxycholate, 0.1% SDS) and sonicated using a Diagenode Bioruptor for three 10-minute rounds using pulsing settings (30 sec ON; 1 min OFF). 10 ug of sonicated chromatin was then incubated overnight at 4oC with 5 ug of antibodies-conjugated to magnetic beads.  Following the IP the beads were washed twice with RIPA buffer (50mM Tris-HCl pH8, 150 mM NaCl, 2mM EDTA, 1% NP-40, 0.1% Na-deocycholate, 0.1% SDS), low salt buffer (20mM Tris pH 8.1, 150mM NaCl, 2mM EDTA, 1% Triton X-100, 0.1% SDS), high salt buffer (20mM Tris pH 8.1, 500mM NaCl, 2mM EDTA, 1% Triton X-100, 0.1% SDS), LiCl buffer (10mM Tris pH 8.1, 250mM LiCl, 1mM EDTA, 1% Na-deoxycholate, 1% NP-40), and 1xTE. Finally, DNA was extracted by reverse crosslinking at 60oC overnight with proteinase K (20ug/ul) and 1% SDS followed by phenol:chloroform:iso-amylacohol purification. Libraries were constructed as indicated below and sequenced using paired-end 50 bp sequencing reactions
TruSeq illumina barcodes were ligated to immunoprecipitated DNA following end-repair. Minimal PCR amplification of 7-9 cycles produced suffienct material for sequening on a NovaSeq S2.

value: ChIP-seq

value: Input


title: Human male naive hESC WIN8 - H3K9AC

description: ChIP-seq using H3K9ac antibody in female navie hESCs (WIN1)

value: H3K9ac antibody


title: Human male naive hESC WIN4 - H3K4ME1

description: ChIP-seq using H3K4me1 antibody in female navie hESCs (WIN1)

value: H3K4me1 antibody


title: Human male naive hESC WIN6 - H3K4ME3

description: ChIP-seq using H3K4me3 antibody in female navie hESCs (WIN1)

value: H3K4me3 antibody


title: Human male naive hESC WIN9 - H3K9ME3

description: ChIP-seq using H3K9me3 antibody in female navie hESCs (WIN1)

value: H3K9me3 antibody


title: H9_WT_H3K27me3_R2

description: CUT&Tag using H3K27me3 antibody in WT H9 (Rep2)


title: Human male naive hESC WIN3 - H3K36ME3

description: ChIP-seq using H3K36me3 antibody in female navie hESCs (WIN1)

value: H3K36me3 antibody


title: Human male naive hESC WIN2 - H3K27ME3

description: ChIP-seq using H3K27me3 antibody in female navie hESCs (WIN1)

value: H3K27me3 antibody


title: Human Female naive iPSC - H3K9ME3

description: ChIP-seq using H3K9me3 antibody in female navie iPSCs

source_name: iPSC

value: Naive iPSCs


title: Human male naive hESC WIN7 - H3K79ME2

description: ChIP-seq using H3K79me2 antibody in female navie hESCs (WIN1)

value: Naive hESC

value: H3K79me2 antibody


title: Human Female naive iPSC - INPUT

description: ChIP-seq input (used as control) in female navie iPSCs


title: Human male naive hESC WIN1 - H3K27AC

description: ChIP-seq using H3K27ac antibody in female navie hESCs (WIN1)

value: H3K27ac antibody


title: Human Female naive iPSC - H3K79ME2

description: ChIP-seq using H3K79me2 antibody in female navie iPSCs


title: Human Female naive iPSC - H3K9AC

description: ChIP-seq using H3K9ac antibody in female navie iPSCs


title: Human Female naive iPSC - H3K4ME2

description: ChIP-seq using H3K4me2 antibody in female navie iPSCs

value: H3K4me2 antibody


title: Human Female naive iPSC - H3K4ME3

description: ChIP-seq using H3K4me3 antibody in female navie iPSCs


title: Human Female naive iPSC - H3K4ME1

description: ChIP-seq using H3K4me1 antibody in female navie iPSCs


title: Human Female naive iPSC - H3K27ME3

description: ChIP-seq using H3K27me3 antibody in female navie iPSCs


title: Human Female naive iPSC - H3K36ME3

description: ChIP-seq using H3K36me3 antibody in female navie iPSCs


title: H9 XIST KO (C7)

description: Single cell RNA of XIST KO in Naive hESCs (H9) (Clone7)

data_processing:
Align: CellRanger (v3.0.2) count function was used against hg38 (GRCh38-3) genome assembly. To account for mouse feeder cells, we also aligned the reads against the mouse mm10 genome assembly.
Process: Seurat (v3.1.5) was used for further processing and filtering based on multiple criteria: 1) Initial filtering for genes detected in at least three cells, and cells where at least 800 detected genes. 2) Filtering for cells classified as human by CellRanger GEM classification, to remove mouse feeder cells. 3) Mitochondrial RNA was quantified per cell. The R function isOutlier (scater package v1.12.2) was used to identify outliers based on the library size (nmads>3), number of genes (nmads>3), and the percentage of mitochondrial genes in each cell (nmads>1)
Normalize and scale: sctransform function (Seurat package (v3.1.5)) using default parameters
Cluster: Principal Component Analysis was done using Seurat (v3.1.5) function RunPCA on the most highly variable genes. The top 20 principal components were used to find the 20 nearest neighbors of each cell (using FindNeighbors function from Seurat package), following by FindClusters function to identify cell clusters. Clusters were visualized by uniform manifold approximation and projection (UMAP) dimensional reduction technique using the Seurat RunUMAP function.
Assembly: hg38
Supplementary files format and content: Matrix table with raw gene counts for every single cell

growth_protocol: Cells used for in this experiment were cultured in 5iLAF naïve ESC medium (1:1 mixture of DMEM/F12 and Neurobasal supplemented with N2, B27, penicillin/streptomycin, nonessential amino acids, GlutaMAX, 0.5% KSR, 0.1mM β-mercaptoethanol, 50ug/ml bovine serum albumin, 20ng/ml _x005F_x005F_x005F_x000B_rhLIF, 20ng/ml Activin A, 8ng/ml FGF2, 1uM MEK inhibitor _x005F_x005F_x005F_x000B_PD0325901 0.5uM B-Raf inhibitor SB590885, 1uM GSK3-β inhibitor IM-12, 1uM Src inhibitor WH-4-023, and _x005F_x005F_x005F_x000B_10uM ROCK inhibitor Y-27632). Both H9 WT and XIST deletion hESCs were cultured in 5% CO2, 5% O2 (Hypoxia) at 37C.

extract_protocol: scRNA-seq libraries were generated immediately after cell dissociation using 10X Genomics Chromium instrument and Chromium single cell 3’ reagent kit V3. Each individual run was designed to target 10 000 cells per library. Libraries were generated according to manufacturer’s instructions and library fragment size distribution was determined by BioAnalyzer instrument. Afterwards libraries were pulled together and sequenced on Illumina Novaseq 6000 platform.

treatment_protocol: Prior to dissociation cells were washed with PBS and afterwards cells were dissociated with Accutase for 5 minutes at 37C. Afterwards cells were resuspended in MEF media and span down at 1000 rpm for 5 minutes. Cell pallet was washed ones with  PBS+0.04% BSA and after 5 minute spin cells were resuspended in PBS+0.04% BSA. To get rid of cell clusters, suspension was strained through 40 micron strainer. Prior to 10X run cell number and viability was assessed by Countess automated cell counter (Thermo).

value: Naive HESCs

value: scRNA-seq

value: XIST KO


title: H9 XIST KO (C18)

description: Single cell RNA of XIST KO in Naive hESCs (H9) (Clone18)


title: Human Female prime iPSC – Input

description: Input DNA in prime iPSCs

data_processing:
Trim: trim_galore (v0.4.1) with default parameters was used to trim DNA sequencing reads
Align: Bowtie2 (v2.2.9) with the default parameters was used to align reads
Filter: Samtools (v1.9) was used to remove reads with mapping quality less than 30, or marked by Picard MarkDuplicates (v2.1.0) as PCR duplicates
Count: Bedtools (v2.26.0) intersect was used to count reads in each genomic region (either 100kb windows every 25kb, 1Mb every 250kb, or around transcription start site)
Enrichment: read counts in each window were normalized by the total read counts in each sample. The ratio of the normalized reads in the pulldown to the input were used as inputs
Peaks: MACS2 (v2.2.7.1) using broad and max-gap=100 was used to call peaks
Assembly: hg38
Supplementary files format and content: bedgraph files represent normalized enrichment ratios in windows across the genome (either 100kb windows every 25kb, 1Mb every 250kb, or around gene TSS). Is_unmappable=yes mark unmappable genomic regions
Supplementary files format and content: peak files represent broad peaks called for XIST pulldown files samples compared to input
Library strategy: RAP-seq

growth_protocol: Naïve hESCs (UCLA1 and H9) and iPSCs were cultured in 5iLAF naïve ESC medium on irradiated male mouse embryonic fibroblasts (1:1 mixture of DMEM/F12 and Neurobasal supplemented with N2, B27, penicillin/streptomycin, nonessential amino acids, GlutaMAX, 0.5% KSR, 0.1mM β-mercaptoethanol, 50ug/ml bovine serum albumin, 20ng/ml rhLIF, 20ng/ml Activin A, 8ng/ml FGF2, 1uM MEK inhibitor PD0325901 0.5uM B-Raf inhibitor SB590885, 1uM GSK3-β inhibitor IM-12, 1uM Src inhibitor WH-4-023, and 10uM ROCK inhibitor Y-27632). NHDFs were cultured in DMEM, with 10% FBS, penicillin/streptomycin, nonessential amino acids, GlutaMAX and 0.1mM β-mercaptoethanol. H9 hESCs were cultured in 5% CO2, 5% O2 (Hypoxia) at 37C and UCLA1, iPSCs and NHDFs were cultured in normal atmospheric O2 and 5% CO2 at 37C.

extract_protocol:
After washing with PBS, 5-30 million cells were harvested from confluent cell cultures and incubated with freshly-made 10ml 2mM DSG in 1x PBS at room temperature for 45 minutes. Cells were subsequently crosslinked further at room temperature with 10ml 3% formaldehyde for 10 minutes, and the reaction was stopped by the addition of 2ml 2.5M glycine. Cells were pelleted at 4˚C, aliquoted into vials of 10 million cells, and subjected to lysate preparation as described by Engreitz et al. (Engreitz et al., 2013). Briefly, the cells were passively lysed in Cell Lysis Buffer (10mM HEPES at pH7.5, 20mM KCl, 2mM MgCl2, 1mM EDTA with 0.1% NP-40) for 10min on ice, then lysis was completed either with glass dounce homogenizer (UCLA1, naïve iPSCs and NHDFs) or without (naïve (WT and with XIST KO) and primed H9, and primed iPSCs). Nuclei were lysed in Nuclear Lysis Buffer (20mM HEPES at pH7.5, 50mM KCl, 1.5mM MnCl2, 1% NP-40, 0.4% Sodium deoxycholate, 0.1% N-lauroylsarcosine) for 10min on ice. Chromatin was solubilized by sonication. Briefly, for UCLA1, naïve iPSCs and NHDFs, five cycles of sonication were performed, with each cycle lasting 45 seconds on high power. For naïve and primed H9, and primed iPSCs sonication was done with a Misonix S-400 sonicator with microtip (model number: U1240A0418) at an amplitude of 12, total process time of 2 min with pulse on for 1sec and pulse off for 3sec. After sonication, chromatin was further segmented by incubating it with TURBO DNase digestion at a concentration of 0.1-0.4U/ul for 12-20min at 37C (time was optimized for each sample). The digestion was stop by the addition of DNase Stop Solution (mixture of EDTA and EGTA). XIST RNA pulldown from 5 million cells was done using 1ug (UCLA1, iPSCs, NHDF R1) or 50pmol (primed and naïve H9 and primed iPSCs) of non-overlapping 90nt long biotinylated oligonucleotide probes (Eurofins) (Table S3). To test the effect of higher concentration of the biotinylated oligonucleotides, a higher concentration was added to one replicate of NHDF (R2), using 5ug probes. 1mg (UCLA1, naïve iPSCs and NHDFs) or 0.6mg (naïve and primed H9, and primed iPSCs) of C1 Streptavidin beads were used. As a control, 5% of each sample was taken as an input control and the remaining sample was used for the pulldown. DNA was eluted by Rnase H digestion, and crosslinking was reversed via proteinase K digestion of eluted DNA at 60˚C 10-12 hours.
DNA libraries were prepared using NEBNext Ultra End Rpair/dA-Tailing Module (NEB) and TruSeq DNA adapters (Illumina) ligated using Quick Ligase (NEB). Libraries were amplified by KAPA HiFi Polymerase (Roche), pooled, and sequenced on the Illlumina HiSeq platform to generate 50bp single-end reads (UCLA1, naïve iPSCs and NHDFs) or pair-end reads (naïve and primed H9 and primed iPSCs).

value: iPSC

value: Prime iPSCs

value: RAP-Seq


title: Human Female prime iPSC – XIST Pulldown

description: RAP-Seq using probes targeting Xist in prime iPSCs

value: XIST RAP


title: H9 WT (Cl18)

description: Single cell RNA of Naive hESCs (H9) used as control for XIST KO (Clone18)

value: WT


title: H9 WT (C7)

description: Single cell RNA of Naive hESCs (H9) used as control for XIST KO (Clone7)


title: Human male naive hESC WIN5 - H3K4ME2

description: ChIP-seq using H3K4me2 antibody in female navie hESCs (WIN1)

value: ChIP-seq


title: Human Female naive iPSC - H3K27AC

description: ChIP-seq using H3K27ac antibody in female navie iPSCs


title: Human Female prime hESC H9 – Input

description: Input DNA in prime hESCs (H9)

value: Prime hESC

value: Input


title: Human Female naive hESC H9 with XIST KO – Input

description: Input DNA in naive hESCs (H9) with XIST KO


title: Human Female prime hESC H9 – XIST Pulldown

description: RAP-Seq using probes targeting Xist in prime hESCs (H9)


title: Female naïve hESCs (H9) with XIST KO (C7) in siSPEN R1

description: 5iLAF-cultured, derived from primed hESCs at passage 43, p21 in 5iLAF, with siSPEN treatment for 48 hours, replicate 1

data_processing:
Trim: RNA sequencing reads were trimmed using trim_galore (v0.4.1) with default parameters
Align: HISAT2 (v2.2.0) with default parameters was used to align RNA reads
Filter: Samtools (v1.9) was used to remove reads with mapping quality less than 30
Counts: HTSeq (v.0.6.1p1) with the following parameters “--format=bam --order=pos --stranded=reverse--minaqual=0 --type=exon --mode=union –idattr=gene_name" was used to calculate read counts overlapping with genes
Assembly: hg38
Supplementary files format and content: Matrix table with raw gene counts for every gene and every sample

molecule: polyA RNA

growth_protocol: All cells except HNES lines were cultured in 5iLAF naïve ESC medium (1:1 mixture of DMEM/F12 and Neurobasal supplemented with N2, B27, penicillin/streptomycin, nonessential amino acids, GlutaMAX, 0.5% KSR, 0.1mM β-mercaptoethanol, 50ug/ml bovine serum albumin, 20ng/ml rhLIF, 20ng/ml Activin A, 8ng/ml FGF2, 1uM MEK inhibitor PD0325901 0.5uM B-Raf inhibitor SB590885, 1uM GSK3-β inhibitor IM-12, 1uM Src inhibitor WH-4-023, and 10uM ROCK inhibitor Y-27632) and were cultured either in 5% CO2, 5% O2 (Hypoxia) at 37C or in 5% CO2 and atmospheric oxygen level (Normoxia) at 37C. HNES1 and HNES3 were cultured in naive ESC medium consisting of (1:1 mixture of DMEM/F12 and Neurobasal supplemented with N2, B27, penicillin/streptomycin, nonessential amino acids, GlutaMAX, 0.5% KSR, 0.1mM β-mercaptoethanol, 50ug/ml bovine serum albumin, 20ng/ml rhLIF, 2uM XAV939, 2uM Go6983, 1uM MEK inhibitor PD0325901, and 10uM ROCK inhibitor Y-27632) and were cultured in 5% CO2, 5% O2 at 37C)

extract_protocol:
RNA was isolated using Qiagen RNAeasy kit (#74104) according to manufacturer’s instructions
Libraries were prepared with the TruSeq Stranded mRNA Library Prep Kit (Illumina 20020594) according to manufacturer’s instructions. Library quality was assessed by TapeStation (Agilent) and subsequently quantified using the Qubit dsDNA High-Sensitivity Kit (Life Technologies)

treatment_protocol: Cells were washed with 1xPBS and dissociated with accutase for 5 minutes at 37C. Afterwards cells were resuspended in MEF media and span down at 1000 rpm for 5 minutes. Cell pallet was washed once with PBS+0.04% BSA and after 5 minute spin cell pallet was resuspended in Trizol reagent (Life Technologies #15596018) for RNA isolation

value: naive hPSCs

value: RNA-seq

value: siSPEN in XIST KO

tag: origin

value: This study

tag: culture condition

value: Hypoxia


title: Female naïve hESCs (H9) with XIST KO (C7) in siSPEN R2

description: 5iLAF-cultured, derived from primed hESCs at passage 43, p21 in 5iLAF, with siSPEN treatment for 48 hours, replicate 2


title: Female naïve hESCs (H9) with XIST KO (C18) in siSPEN R1


title: Female naïve hESCs (H9) with XIST KO (C7) in siSPEN control R1

description: 5iLAF-cultured, derived from primed hESCs at passage 43, p21 in 5iLAF, with siCtrl treatment for 48 hours, replicate 1


title: Human Female naive hESC H9 – Input

description: Input DNA in naive hESCs (H9)

value: Naive hESC


title: Female naïve hESCs (H9) with XIST KO (C18) in siSPEN R2


title: Female naïve hESCs (H9) with XIST KO (C7) in siSPEN control R2

description: 5iLAF-cultured, derived from primed hESCs at passage 43, p21 in 5iLAF, with siCtrl treatment for 48 hours, replicate 2


title: Male naïve siSPEN (WIN1) R2

description: 5iLAF-cultured, derived from a blastocyst, with siSPEN treatment for 48 hours, passage 28, replicate 2

value: siSPEN

value: Normoxia


title: Female naïve hESCs (H9) with XIST KO (C18) in siSPEN control R2

value: XIST KO


title: Female naïve hESCs (H9) with XIST KO (C18) in siSPEN control R1


title: Male siSPEN control (WIN1)

description: 5iLAF-cultured, derived from a blastocyst, with siCtrl treatment for 48 hours, passage 28, replicate 2


title: Male naïve siSPEN (WIN1) R1

description: 5iLAF-cultured, derived from a blastocyst, with siSPEN treatment for 48 hours, passage 28, replicate 1


title: Female siSPEN control (UCLA1)

description: 5iLAF-cultured, derived from the primed state, with siCtrl treatment for 48 hours, passage 24


title: Female naïve siSPEN (UCLA1) R1

description: 5iLAF-cultured, derived from the primed state, with siSPEN treatment for 48 hours, passage 24, replicate 1


title: Female naïve siSPEN (UCLA1) R2

description: 5iLAF-cultured, derived from the primed state, with siSPEN treatment for 48 hours, passage 24, replicate 2


title: Male naïve ESCs (WIN1) R2

description: 5iLAF-cultured, derived from a blastocyst, passage 25, replicate 2


title: Male naïve HNES1 R2

description: t2iL+Gö-cultured, derived from blastocyst, replicate 2


title: Male naïve ESCs (WIN1) R1

description: 5iLAF-cultured, derived from a blastocyst, passage 25, replicate 1


title: Female naïve iPSC R2

description: 5iLAF-cultured, derived from human fibroblasts, passage 11 in 5iLAF after subcloning, replicate 2

value: iPSC

value: naive iPSCs


title: Female naïve iPSC R1

description: 5iLAF-cultured, derived from human fibroblasts, passage 11 in 5iLAF after subcloning, replicate 1


title: Female naïve HNES3 R2


title: Female naïve hESCs (H9) with XIST KO (C7) R10

description: 5iLAF-cultured, derived from primed hESCs at passage 43, p19 in 5iLAF, replicate 2

value: naive hESCs


title: Female naïve hESCs (H9) with XIST KO (C7) R11

description: 5iLAF-cultured, derived from primed hESCs at passage 43, p19 in 5iLAF, replicate 3


title: Human Female naive hESC H9 with XIST KO – Pulldown

description: RAP-Seq using probes targeting Xist in naive hESCs (H9) with XIST KO

value: RAP-Seq

value: XIST RAP


title: Human Female naive hESC H9 – XIST Pulldown

description: RAP-Seq using probes targeting Xist in naive hESCs (H9)


title: Female naïve hESCs (H9) with XIST KO (C7) R8

description: 5iLAF-cultured, derived from primed hESCs at passage 43, p15 in 5iLAF, replicate 3


title: Female naïve hESCs (H9) with XIST KO (C7) R9

description: 5iLAF-cultured, derived from primed hESCs at passage 43, p19 in 5iLAF, replicate 1


title: Female naïve hESCs (H9) with XIST KO (C7) R7

description: 5iLAF-cultured, derived from primed hESCs at passage 43, p15 in 5iLAF, replicate 2


title: Female naïve hESCs (H9) with XIST KO (C7) R6

description: 5iLAF-cultured, derived from primed hESCs at passage 43, p15 in 5iLAF, replicate 1


title: Female naïve hESCs (H9) with XIST KO (C7) R4

description: 5iLAF-cultured, derived from primed hESCs at passage 42, p8 in 5iLAF, replicate 2


title: Female naïve hESCs (H9) with XIST KO (C7) R2

description: 5iLAF-cultured, derived from primed hESCs at passage 46, p8 in 5iLAF, replicate 2


title: Female naïve hESCs (H9) with XIST KO (C7) R5

description: 5iLAF-cultured, derived from primed hESCs at passage 42, p8 in 5iLAF, replicate 3


title: Male naïve HNES1 R1

description: t2iL+Gö-cultured, derived from blastocyst, replicate 1

value: WT


title: Female naïve hESCs (H9) with XIST KO (C7) R1

description: 5iLAF-cultured, derived from primed hESCs at passage 46, p8 in 5iLAF, replicate 1


title: Female naïve hESCs (H9) with XIST KO (C7) R3

description: 5iLAF-cultured, derived from primed hESCs at passage 42, p8 in 5iLAF, replicate 1


title: Female naïve hESCs (H9) with XIST KO (C18) R5

description: 5iLAF-cultured, derived from primed hESCs at passage 50, p8 in 5iLAF, replicate 3


title: Female naïve hESCs (H9) with XIST KO (C18) R3

description: 5iLAF-cultured, derived from primed hESCs at passage 50, p8 in 5iLAF, replicate 1


title: Female naïve hESCs (H9) with XIST KO (C18) R9

description: 5iLAF-cultured, derived from primed hESCs at passage 47, p19 in 5iLAF, replicate 1


title: Female naïve hESCs (H9) with XIST KO (C18) R7

description: 5iLAF-cultured, derived from primed hESCs at passage 47, p15 in 5iLAF, replicate 2


title: Female naïve hESCs (H9) with XIST KO (C18) R11

description: 5iLAF-cultured, derived from primed hESCs at passage 47, p19 in 5iLAF, replicate 3


title: Female naïve hESCs (H9) with XIST KO (C18) R4

description: 5iLAF-cultured, derived from primed hESCs at passage 50, p8 in 5iLAF, replicate 2


title: Female naïve hESCs (H9) with XIST KO (C18) R6

description: 5iLAF-cultured, derived from primed hESCs at passage 47, p15 in 5iLAF, replicate 1


title: Female naïve hESCs (H9) with XIST KO (C18) R1


title: Female naïve ESCs (UCLA1) R4

description: 5iLAF-cultured, derived from the primed state, subclone 12, passage 48, replicate 4


title: Female naïve hESCs (H9) with XIST KO (C18) R2


title: Female naïve HNES3 R1


title: Female naïve hESCs (H9) with XIST KO (C18) R10

description: 5iLAF-cultured, derived from primed hESCs at passage 47, p19 in 5iLAF, replicate 2


title: Female naïve ESCs (H9) R10

description: 5iLAF-cultured, derived from primed H9 hESCs at passage 51, p17 in 5iLAF, replicate 3


title: Female naïve ESCs (UCLA1) R3

description: 5iLAF-cultured, derived from the primed state, subclone 12, passage 48, replicate 3


title: Female naïve ESCs (H9) R8

description: 5iLAF-cultured, derived from primed H9 hESCs at passage 51, p17 in 5iLAF, replicate 1


title: Female naïve ESCs (H9) R9

description: 5iLAF-cultured, derived from primed H9 hESCs at passage 51, p17 in 5iLAF, replicate 2


title: Female naïve ESCs (H9) R5


title: Female naïve ESCs (H9) R7


title: Female naïve ESCs (H9) R6


title: Female naïve ESCs (H9) R4

description: 5iLAF-cultured, derived from primed H9 hESCs at passage 48, p9 in 5iLAF, replicate 2


title: Female naïve ESCs (H9) R3

description: 5iLAF-cultured, derived from primed H9 hESCs at passage 48, p9 in 5iLAF, replicate 1


title: Female naïve hESCs (H9) with XIST KO (C18) R8

description: 5iLAF-cultured, derived from primed hESCs at passage 47, p15 in 5iLAF, replicate 3


title: Female naïve ESCs (H9) R2

description: 5iLAF-cultured, derived from primed H9 hESCs at passage 46, p8 in 5iLAF, replicate 2


title: GBM_3.7.T

city: Melbourne

first: Joel

middle: Jeffrey Duncan

last: Moffet

state: Victoria

address: 1G Royal Parade

country: Australia

institute: WEHI

data_processing:
Raw counts from the two separate experimental batches were grouped into a single dataset. AOIs were removed if their nuclei count was below 100 nuclei, as provided by NanoString. Genes were filtered out unless above limit of quantitation in 5 % of remaining AOIs, calculated for each AOI as the geometric mean of all negative probes multiplied by the geometric standard deviations of the negative probes to the second power. Remaining genes were normalized by upper quartile normalization
Assembly: hg38
Supplementary files format and content: tab-delimited text file containing matrix of upper quartile normalized gene counts for each sample

source_name: primary high grade glioma

extract_protocol:
Formalin fixed paraffin embedded (FFPE) tissue were sectioned at 5 μm thickness and mounted onto Superfrost Plus slides within one month of spatial transcriptomics analysis. Slides were stored at 4 degrees Celsius with desiccator and shipped to NanoString (Seattle, USA) for Technology Access Program (TAP) processing for GeoMx DSP.
Pathology review was performed on all the six cases. The tissue sections demonstrated distinct morphological features and were assessed using the cellularity of the tumor cells, nuclear pleomorphisms, presence of mitoses, necrosis, and microvascular proliferation. The five anatomic features proposed by The Ivy Glioblastoma Atlas Project (Ivy GAP) were used to annotate the tumors into regions. These included: Leading edge (LE): the outermost part of tumour with the least tumor cell density and bordering on the normal tissue area; Cellular tumor (CT): the core of the tumor with highest cell density; Infiltrating tumor (IT): the zone interposed between LE and CT with moderate tumour cell density; Microvascular proliferation (MVP): Areas with endothelial proliferation; Pseudopalisading cells around necrosis (PAN): dense tumour cells geographically fenced around core necrosis. Other features recognized are regions of large non-palisaded necrosis (NEC) and normal brain tissue (N). Annotations were performed by two pathologists.
After baking the slides for 2 hours for paraffin removal, slides were loaded onto a Leica BOND RX for tissue rehydration, heat-induced epitope retrieval (ER2 for 20 minutes at 100 °C) and proteinase K treatment (0.1 μg/ml for 15 minutes at 37 °C). The tissue sections were then hybridized with the Human WTA probes overnight. Following 2 x 5 min stringent washes (1:1 4x SSC buffer & formamide), the slides were blocked and then incubated morphology marker antibodies: GFAP (488 channel, NBP2-3318AF488, Novus), CD45 (594 channel, 13917BF, Cell Signaling Technologies), and Ki67 (647 channel, 9027BF, Cell Signaling Technologies). Syto83 (532 channel, S11364, Invitrogen) was used as a nuclear stain. Tissue sections were then loaded into the GeoMx platform and Regions of interest (ROIs) were selected guided by the immunofluorescence of the morphology markers antibodies listed above. Segmentation was used in some ROIs used to enrich for Ki67+ and/or CD45+ populations. UV light was then directed by the GeoMx at each AOI and released the RNA ID and UMI-containing oligonucleotide tags from the WTA probes for collection and sequencing preparation. Illumina i5 and i7 dual indexing primers were added to the oligonucleotide tags during PCR to uniquely index each AOI. AMPure XP beads (Beckman Coulter) were used for PCR purification. Library concentration as measured using a Qubit fluorometer (Thermo Fisher Scientific) and quality was assessed using a Bioanalyzer (Agilent). Sequencing was performed on an Illumina NovaSeq 6000 and .fastq files were processed into gene count data for each AOI using the GeoMx® NGS Pipeline.

value: glioblastoma

tag: sample

tag: id simple

tag: region

tag: id sample

tag: class

value: GBM

tag: Sex

value: M

tag: lib size

tag: immune border

tag: aoinucleicount

tag: classification

value: Border

tag: infiltration

value: Low

tag: ivygap

value: Cellular Tumour

value: positive

tag: roi type

value: tumor

tag: age

tag: tumor location

value: Left temporal

value: Intact

tag: atrx

value: Mosaic

value: Mutated

tag: ki67 percentage

tag: cytology

tag: who grade

tag: comments

value: MGMT unmethylated

tag: loq


title: Female naïve ESCs (H9) Normoxia R2

tag: cell line

tag: cell type

value: naive hESCs

tag: library strategy

value: RNA-seq

tag: genotype

tag: origin

value: This study

tag: culture condition

value: Normoxia


title: Female naïve ESCs (H9) Normoxia R1

description: 5iLAF-cultured, derived from primed H9 hESCs at passage 46, p8 in 5iLAF, replicate 1

value: WT


title: GBM_3.6.T

tag: tissue

value: glioblastoma

tag: sample

tag: id simple

tag: region

tag: id sample

tag: class

value: GBM

tag: Sex

tag: batch

tag: lib size

tag: immune border

tag: aoinucleicount

tag: classification

value: Normal

tag: infiltration

value: High

value: Necrosis

value: non


title: GBM_3.7.I

value: M

value: Border

value: Low

tag: ivygap

value: Cellular Tumour

tag: roi type

tag: age

tag: tumor location

tag: atrx

tag: ki67 percentage

tag: cytology

tag: who grade

tag: comments

tag: loq


title: GBM_3.6.I

tag: tissue

tag: sample

tag: id simple

tag: region

tag: id sample

tag: class

tag: Sex

value: Left temporal

value: Intact

value: Mosaic

value: Mutated


title: GBM_3.4.T

tag: batch

tag: lib size

tag: immune border

tag: aoinucleicount

tag: classification

value: Ki67- Border

tag: infiltration

tag: ivygap

value: Pseudo-palisading cells Around the Necrosis

value: negative

value: tumor

value: MGMT unmethylated


title: GBM_3.5.T

value: glioblastoma

value: GBM

value: positive

tag: roi type

tag: age

tag: tumor location

tag: atrx

tag: ki67 percentage

tag: cytology

tag: who grade


title: GBM_3.4.I

tag: tissue

tag: sample

tag: id simple

tag: region

tag: id sample

value: High

value: non

tag: comments

tag: loq


title: GBM_3.5.I

tag: class

tag: Sex

value: M

tag: batch

tag: lib size

tag: immune border

tag: aoinucleicount

tag: classification

value: Border


title: GBM_3.1.T

tag: infiltration

value: Low

tag: ivygap

tag: roi type

tag: age

tag: tumor location

tag: atrx

tag: ki67 percentage


title: GBM_3.3.I

tag: tissue

tag: sample

tag: id simple

tag: region

tag: id sample

value: Perinecrotic Zone

value: Left temporal

tag: cytology

tag: who grade


title: GBM_3.3.T

tag: class

tag: Sex

tag: batch

tag: lib size

tag: immune border

value: Intact

value: Mosaic

tag: comments

tag: loq


title: GBM_3.1.I

tag: aoinucleicount

tag: classification

value: Ki67- Border

tag: infiltration

tag: ivygap

tag: roi type

tag: age

tag: tumor location

value: Mutated


title: GBM_3.2.T

tag: tissue

tag: sample

tag: id simple

tag: region

tag: id sample

value: Cellular Tumour

tag: atrx

tag: ki67 percentage


title: GBM_3.2.I

tag: class

tag: Sex

tag: batch

tag: lib size

tag: immune border

tag: cytology

tag: who grade


title: GBM_2.6.T

tag: aoinucleicount

tag: classification

tag: infiltration

tag: ivygap

value: negative

tag: roi type

value: tumor

value: Left frontal

tag: comments

tag: loq


title: Female naïve ESCs (H9) R1

tag: cell line

tag: cell type

value: naive hESCs

tag: library strategy

value: RNA-seq

tag: genotype

value: WT

tag: origin

value: This study

tag: culture condition

value: Hypoxia


title: GBM_2.3.I

tag: tissue

value: glioblastoma

tag: sample

tag: id simple

tag: region

tag: id sample

tag: class

value: GBM

tag: Sex

value: M

tag: batch

tag: lib size

value: Infiltrating Tumour

value: non

tag: age

tag: tumor location

tag: atrx

tag: ki67 percentage

value: MGMT unmethylated


title: GBM_2.3.T

tag: immune border

tag: aoinucleicount

tag: classification

value: Ki67- Border

tag: infiltration

value: Low

tag: ivygap

tag: roi type

value: Intact

tag: cytology

tag: who grade

tag: comments

tag: loq


title: GBM_2.4.T

tag: tissue

tag: sample

tag: id simple

tag: region

tag: id sample

tag: class

tag: Sex

value: Border

value: High

value: Cellular Tumour

value: positive

value: Mutated


title: GBM_2.6.I

tag: batch

tag: lib size

tag: immune border

tag: age

tag: tumor location

value: Left frontal

tag: atrx

tag: ki67 percentage


title: GBM_2.5.T

tag: aoinucleicount

tag: classification

tag: infiltration

tag: ivygap

value: negative

tag: roi type

value: tumor

tag: cytology

tag: who grade


title: GBM_2.6.K

tag: tissue

value: glioblastoma

tag: sample

tag: id simple

tag: region

tag: id sample

tag: comments

tag: loq


title: GBM_2.4.I

tag: class

value: GBM

tag: Sex

tag: batch

tag: lib size

tag: immune border

tag: age

tag: tumor location


title: A_3.4.T

value: anaplastic astrocytoma

value: A

value: M

tag: aoinucleicount

tag: classification

tag: infiltration

tag: ivygap

tag: roi type

value: Right frontal

tag: atrx

tag: ki67 percentage

value: Pre-treated:radiation, temozolomide


title: A_3.5.T

tag: tissue

tag: sample

tag: id simple

tag: region

tag: id sample

value: Low

tag: cytology

tag: who grade


title: GBM_2.2.K

tag: class

tag: Sex

tag: batch

tag: lib size

tag: immune border

value: Intact

tag: comments

value: MGMT unmethylated

tag: loq


title: GBM_2.2.T

tag: aoinucleicount

tag: classification

value: Border

tag: infiltration

tag: ivygap

tag: roi type

tag: age

tag: tumor location


title: GBM_2.2.I

tag: tissue

tag: sample

tag: id simple

tag: region

tag: id sample

value: Cellular Tumour

value: non

tag: atrx

tag: ki67 percentage


title: A_3.3.K

tag: class

tag: Sex

tag: batch

tag: lib size

tag: immune border

value: High

value: Infiltrating Tumour

value: positive

value: Mutated

tag: cytology

tag: who grade


title: A_3.2.T

tag: aoinucleicount

tag: classification

tag: infiltration

tag: ivygap

value: Tumour Leading Edge

value: negative

tag: roi type

value: tumor

tag: comments

tag: loq


title: GBM_2.5.I

tag: tissue

value: glioblastoma

tag: sample

tag: id simple

tag: region

tag: id sample

value: GBM

value: Ki67- Border

tag: age

tag: tumor location

value: Left frontal


title: A_3.3.T

value: anaplastic astrocytoma

tag: class

value: A

tag: Sex

value: M

tag: batch

tag: lib size

tag: immune border

value: Right frontal

tag: atrx

tag: ki67 percentage

value: Pre-treated:radiation, temozolomide


title: 3543_OF1A_6-4-I2-P19

tag: brain region

value: Dorsolateral Prefrontal cortex (Brodmann area 46)

value: Female

tag: molecule subtype

value: nuclear RNA + genomic DNA


title: 3543_OF1A_6-4-I2-O20


title: 3543_OF1A_6-4-I2-O8


title: 3543_OF1A_6-4-I2-O7


title: 3543_OF1A_6-4-I2-M7


title: 3543_OF1A_6-4-I2-N8


title: 3543_OF1A_6-4-I2-N20


title: 3543_OF1A_6-4-I2-M20


title: 3543_OF1A_6-4-I2-N19


title: 3543_OF1A_6-4-I2-M8


title: A_3.4.K

tag: aoinucleicount

tag: classification

value: Border

tag: infiltration

value: High

tag: ivygap

value: Infiltrating Tumour

value: positive

tag: roi type

tag: cytology

tag: who grade

tag: comments

tag: loq


title: 3543_OF1A_6-4-I2-M19

tag: age

tag: Sex


title: 3543_OF1A_6-4-I2-L8


title: 3543_OF1A_6-4-I2-K7


title: 3543_OF1A_6-4-I2-L7


title: 3543_OF1A_6-4-I2-J7


title: 3543_OF1A_6-4-I2-L20


title: 3543_OF1A_6-4-I2-K20


title: 3543_OF1A_6-4-I2-I7


title: 3543_OF1A_6-4-I2-K8


title: 3543_OF1A_6-4-I2-J8


title: 3543_OF1A_6-4-I2-I19


title: 3543_OF1A_6-4-I2-L19


title: 3543_OF1A_6-4-I2-J19


title: 3543_OF1A_6-4-I2-J20


title: 3543_OF1A_6-4-I2-I20


title: 3543_OF1A_6-4-I2-K19


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