title: Human Dermal Fibroblast, healthy control, untreat, C155

description: Library name: SAMPLE 9


title: LR_SN_54

status: Public on Feb 21 2025

type: SRA

city: Worcester

first: Ryan

last: Logan

state: MA

address: 366 Plantation Street

country: USA

institute: University of Massachusetts Chan Medical School

description: Library name: LR_SN_54

library_source: transcriptomic

data_processing:
For Rattus norvegicus samples: Quality control was performed by FastQC, cutadapt was used to trim low quality and over-represented sequences. STAR was used for alignment with ensembl reference (release 104) and RSEM is used for expression calculation.
For Mus musculus samples: Quality control was performed by Fastqc. Perform trimming by trimmomatic, allign the sequence using Hisat2 with Ensembl reference and read count with Hiseq
Assembly: Mus musculus:GRCm38 ; Rattus norvegicus: Rnor_6.0(Ensembl release 104)
Supplementary files format and content: mouse_raw_counts.csv: processed count data for Mus musculus samples.
Supplementary files format and content: rat_raw_counts_all.csv: processed count data for Rattus norvegicus samples.

molecule: total RNA

organism: Rattus norvegicus

source_name: brain

extract_protocol:
RNA extraction was performed from 0.5 mm punches of coronal sections using RNeasy Micro kits (Qiagen, Inc.). Briefly, tissue samples were homogenized in Buffer RLT, RNA lysis buffer, before being centrifuged in a QIAshredder column at full speed for 3 min. The flow-through was mixed with an equal volume of 70% ethanol and transferred to a MinElute spin column before being washed with Buffer RW1, RNA wash buffer, to free membrane-bound RNA. DNA was removed with on column treatment using DNAse I. The flow-through was washed again with Buffer RW1, and two times with Buffer RPE, a secondary RNA wash buffer in ethanol, to remove trace salts. The spin column was cleaned with 80% ethanol and centrifuged at full speed for 5 min to dry the column matrix. Total RNA was eluted in RNAse-free water. Concentration and purity were quantified using a Nanodrop.
Illumina Total RNAseq Paired End 75bp

tag: tissue

value: brain

tag: cell type

value: neuron

tag: strain

value: Heterogeneous Stock

tag: treatment

value: Abstinence

tag: batch

tag: Sex

value: male

tag: region

value: NAc Shell


title: E1A/HrasG12V MEFs, Zmat3-KO 4

city: Stanford

first: Laura

middle: D

last: Attardi

state: CA

address: 269 Campus Drive

institute: Stanford University

description:
Library name: Sample 12
Column name in CPM_all_samples.txt: Z2G

data_processing:
Reads were aligned to the GRCm38 mouse genome using HISAT2 (v 2.0.4; RRID:SCR_015530).
Sorted BAM files were generated using Samtools (v 1.3.1; RRID:SCR_002105).
The number of reads mapping to each gene in the mouse Ensembl database (v 87; RRID:SCR_002344) was counted using HTSeq-count (v 0.6.1; RRID:SCR_011867).
Differential gene expression analysis was performed using DESeq2 (v 1.24.0; RRID:SCR_015687).
Assembly: GRCm38
Supplementary files format and content: tab-delimited text file includes CPM for each sample

molecule: polyA RNA

organism: Mus musculus

source_name: E1A/HrasG12V-expressing murine embryonic fibroblast

growth_protocol: Cells were maintained in DMEM + 10% FBS and incubated at 37°C in a carbon dioxide incubator held at 5% oxygen.

extract_protocol:
RNA was extracted from cells using the RNeasy midi kit (Qiagen, Cat#75144).
NEBNext Ultra II RNA Library Prep by Illumina

tag: genotype


title: Human Dermal Fibroblast, healthy control, treat, C153

city: Suwon-si

first: yeojin

last: Kim

address: 164 World cup-ro

country: South Korea

institute: ajou Univ.

description: Library name: SAMPLE 8

data_processing:
Trimmomatic v0.39: Sequence reads were trimmed for adapter sequences and low-quality bases using Trimmomatic (parameters: sliding window = 4, mean quality = 15, base quality threshold = 3, minimum length = 36 bp).
HISAT2 v2.2.1: Trimmed sequence reads were mapped to the GRCh38 reference genome using HISAT2 (parameters: --dta, mismatch penalty = 2, insertion penalty = 3, deletion penalty = 3, alignment score minimum = 0.8).
StringTie v2.1.4: Read count extraction and transcript assembly were performed using StringTie. Normalization was applied to estimate FPKM (Fragments Per Kilobase of transcript per Million mapped reads) and TPM (Transcripts Per Kilobase Million) values.
DESeq2 v1.30.1: Differentia  expression analysis was performed using DESeq2 with default parameters. Genes with |fold change| ≥ 2 and a p-value < 0.05 were considered differentially expressed.
Assembly: GRCh38
Supplementary files format and content: Excel file includes TPM values for each Sample

organism: Homo sapiens

source_name: patient-derived fibroblast

growth_protocol: Patient-derived dermal fibroblast were matintained in DMEM supplemented with 10% Fetal Bovine Serum(FBS) and antibiotics in humidified atmosphere with 5% CO2 at 37˚C

extract_protocol:
Total RNA was extracted using the RNeasy Plus Mini Kit (Qiagen, Germany) following the manufacturer’s guidelines.
RNA libraries for RNA-seq were prepared using TruSeq Standard mRNA Library Prep Kit following manufacturer's protocols

treatment_protocol: ER stress was induced by treatment of 10nM Thapsigargin for 24h in 70% confluency

tag: disease state

value: healthy control

value: patient-derived fibroblast

value: WT

value: Thapsigargin


title: RNA-seq OP9_DLL4_3

city: Montréal

first: Nathalie

last: Labrecque

state: QC

address: 110, avenue des Pins O

department: Unité de recherche en immunobiologie des lymphocytes T

country: Canada

institute: Institut de recherches cliniques de Montréal

description: Library name: Sample 28

data_processing:
Sequenced reads were trimmed for sequencing adapters and low-quality 3′ bases using Trimmomatic version 0.35 (Bolger et al., 2014) and then aligned to the reference mouse genome version GRCm38 (or mm10 gene annotation from Gencode version M13, based on Ensembl 88) using STAR version 2.5.1b (Dobin et al., 2013). Gene expression was obtained both as read count directly from STAR as well as computed using RSEM (Li and Dewey, 2011) to obtain gene and transcript level expression, either in transcripts per kilobase million or fragments per kilobase per million mapped reads values, for these stranded RNA libraries. DESeq2 version 1.16.1 was then used to normalize gene read counts and compute differential expression between different experimental conditions (Love et al., 2014). Gene Ontology term (GO-term) enrichment analysis of differentially expressed genes was performed using DAVID (Huang et al., 2009) and determination of relevant upstream regulators was done with QIAGEN’s Ingenuity Pathway Analysis (IPA). GSEA enrichment analysis was performed using the Broad Institute’s software and Molecular Signature Database (MSigDB) (https://www.gsea-msigdb.org/gsea/index.jsp) (Subramanian et al., 2005). For experiments evaluating the impact of BATF overexpression, sequences were trimmed with Trimmomatic version 0.39 (Bolger et al., 2014) and aligned to reference genome GRCm38 using STAR version 2.7.8a (Dobin et al., 2013). Gene counts were determined with featureCounts (Liao et al., 2014). Robust principal component analysis (Chen et al., 2020) was used to detect outlier samples and two samples (one KO-empty and one KO-BATF sample) were therefore excluded from differential expression analysis. DESeq2 v1.24.0 was used to determine differential expression (Love et al., 2014).
Assembly: GRCm38
Supplementary files format and content: Read counts (.xlsx)

source_name: Spleen

extract_protocol:
Cells were FACS-sorted directly into TRIzol reagent, frozen at −80°C and RNA was extracted usind a TRIzol protocol.
Library preparation was done with the KAPA RNA HyperPrep PolyA library preparation kit (Roche) using 150 ng of RNA.

value: Spleen

value: Activated OT-I CD8+ T cell

value: in vitro aCD3/aCD28 activation + culture on OP9 fibroblasts

tag: timepoint

value: Day 5

value: Female


title: LR_SN_55

description: Library name: LR_SN_55

value: Intox


title: Human Dermal Fibroblast, healthy control, treat, C155

description: Library name: SAMPLE 10


title: Human Dermal Fibroblast, healthy control, untreat, C153

description: Library name: SAMPLE 7

value: N


title: E1A/HrasG12V MEFs, Zmat3-KO 2

description:
Library name: Sample 10
Column name in CPM_all_samples.txt: Z1G

value: E1A/HrasG12V-expressing murine embryonic fibroblast


title: RNA-seq OP9_DLL4_1

description: Library name: Sample 26

source_name: Lymph nodes

value: Lymph nodes


title: LR_SN_51

description: Library name: LR_SN_51

value: brain

value: neuron

value: Heterogeneous Stock


title: Human Dermal Fibroblast, healthy control, treat, C138

description: Library name: SAMPLE 6


title: RNA-seq OP9_3

description: Library name: Sample 25


title: LR_SN_53

description: Library name: LR_SN_53

value: Abstinence

value: male

value: NAc Shell


title: Human Dermal Fibroblast, healthy control, untreat, C138

description: Library name: SAMPLE 5

value: healthy control

value: patient-derived fibroblast

value: WT


title: RNA-seq OP9_DLL4_2

description: Library name: Sample 27

value: Activated OT-I CD8+ T cell


title: E1A/HrasG12V MEFs, Zmat3-KO 1

description:
Library name: Sample 9
Column name in CPM_all_samples.txt: Z1A


title: LR_SN_50

description: Library name: LR_SN_50


title: LR_SN_52

description: Library name: LR_SN_52


title: RNA-seq OP9_2

description: Library name: Sample 24

value: in vitro aCD3/aCD28 activation + culture on OP9 fibroblasts


title: RNA-seq OP9_1

description: Library name: Sample 23

value: Day 5


title: E1A/HrasG12V MEFs, Cdkn1a-KO 4

description:
Library name: Sample 8
Column name in CPM_all_samples.txt: GP2


title: Human Dermal Fibroblast, healthy control, treat, C124

description: Library name: SAMPLE 4

value: Thapsigargin


title: LR_SN_49

description: Library name: LR_SN_49


title: E1A/HrasG12V MEFs, Zmat3-KO 3

description:
Library name: Sample 11
Column name in CPM_all_samples.txt: Z2A


title: Human Dermal Fibroblast, healthy control, untreat, C124

description: Library name: SAMPLE 3


title: RNA-seq Day3_KO_8

description: Library name: Sample 22

value: Spleen

value: Notch1/2 KO

tag: infection

value: Listeria monocytogenes

value: Day 3

value: Female


title: LR_SN_48

description: Library name: LR_SN_48

value: brain

value: neuron

value: Withdrawal

value: female

value: DLPFC


title: E1A/HrasG12V MEFs, Cdkn1a-KO 3

description:
Library name: Sample 7
Column name in CPM_all_samples.txt: GP1

value: E1A/HrasG12V-expressing murine embryonic fibroblast


title: E1A/HrasG12V MEFs, Cdkn1a-KO 2

description:
Library name: Sample 6
Column name in CPM_all_samples.txt: AP2


title: Human Dermal Fibroblast, healthy control, treat, C121

description: Library name: SAMPLE 2

value: healthy control

value: patient-derived fibroblast


title: RNA-seq Day3_KO_6

description: Library name: Sample 20

value: Activated OT-I CD8+ T cell


title: LR_SN_47

description: Library name: LR_SN_47

value: Heterogeneous Stock


title: LR_SN_45

description: Library name: LR_SN_45


title: Human Dermal Fibroblast, healthy control, untreat, C121

description: Library name: SAMPLE 1

value: WT

value: N


title: E1A/HrasG12V MEFs, Control 3

description:
Library name: Sample 3
Column name in CPM_all_samples.txt: GA1


title: RNA-seq Day3_KO_4

description: Library name: Sample 18

value: Spleen


title: LR_SN_46

description: Library name: LR_SN_46


title: RNA-seq Day3_KO_5

description: Library name: Sample 19

value: Notch1/2 KO


title: Patient_3_rep4

city: BETHESDA

first: Cihan

last: Oguz

state: MD

address: NIHBC 29B BG RM 1NN20, 9000 ROCKVILLE PIKE

institute: NIH

library_source: transcriptomic single cell

data_processing:
Base calling was performed using RTA 3.4.4, demultiplexing was performed using cellranger v6.1.2 (Bcl2fastq 2.20.0), and alignment was performed using cellranger v6.1.2 (STAR 2.7.2a).
Sequenced reads were aligned to the 10x Genomics provided human reference sequence (refdata-gex-GRCh38-2020-A).
Assembly: hg38
Supplementary files format and content: Cell Ranger scRNA-seq count matrices are in the h5 data format (one h5 file per library)

source_name: gastric mucosa

extract_protocol:
High-viability, single-cell dissociation of fresh gastric tissue from three total gastrectomy specimens from three patients was collected for single cell sequencing utilizing the protocol detailed in Samaranayake, Sarah G., et al. "Protocol for isolation of signet ring cells from human gastric mucosa." STAR protocols 4.4 (2023): 102695. (https://doi.org/10.1016/j.xpro.2023.102695)
The sample concentration and viability were assessed using the LunaFx7 fluorescent cell counter and diluted as necessary with PBS+0.04%BSA. The cellular samples were loaded in the lanes according to the 10X Genomics 3’ v3.1 Single Cell User Guide. Cell partitioning completed successfully with uniform emulsion consistency and the reverse transcription PCR was run overnight. All subsequent steps of GEM recovery, cDNA generation, and quality control were performed as described in the 10X Genomics 3’ v3.1 Single Cell User Guide. Libraries were prepared using the Chromium Connect.

value: gastric mucosa

value: gastric epithelial cells


title: LR_SN_44

description: Library name: LR_SN_44


title: Patient_3_rep3


title: Patient_3_rep2


title: E1A/HrasG12V MEFs, Control 2

description:
Library name: Sample 2
Column name in CPM_all_samples.txt: AG2


title: LR_SN_43

description: Library name: LR_SN_43


title: RNA-seq Day3_KO_3

description: Library name: Sample 17

value: Listeria monocytogenes

value: Day 3

value: Female


title: E1A/HrasG12V MEFs, Control 1

description:
Library name: Sample 1
Column name in CPM_all_samples.txt: AG1


title: RNA-seq Day3_KO_2

description: Library name: Sample 16


title: RNA-seq Day3_KO_7

description: Library name: Sample 21


title: Patient_3_rep1


title: E1A/HrasG12V MEFs, Control 4

description:
Library name: Sample 4
Column name in CPM_all_samples.txt: GA2


title: E1A/HrasG12V MEFs, Cdkn1a-KO 1

description:
Library name: Sample 5
Column name in CPM_all_samples.txt: AP1


title: ATAC-seq KO_2

description: Library name: Sample 4

library_source: genomic

data_processing:
The quality of the raw reads was assessed with FASTQC v0.11.8. Trimming was then performed with TRIMMOMATIC v0.36 (Bolger et al., 2014). Reads were aligned to the mouse reference genome mm10 with BOWTIE2 v2.2.6 (Langmead and Salzberg, 2012) with a mean of 57% of reads uniquely mapped. Alignments were post-processed to remove PCR duplicates (Picard tool v2.4.1) and reads mapping to mitochondrial DNA (samtools v1.8). In order to represent the real Tn5 transposase binding sites of 9 bp, the coordinates of the reads were shifted by 4 bp for the plus strand and by 5 bp for the minus strand, using Deeptools v3.0.1 (Ramírez et al., 2016). The former was also used to remove ENCODE’s blacklisted regions (signal artifact regions) and convert Bam files to BEDPE format. MACS2 was used to identify significant peaks (Zhang et al., 2008). Diffbind v2.10.0 R package was used to generate the count matrix of Tn5 insertion site numbers for each consensus peak (peaks that were present in all samples for each condition). DARs were identified between WT vs Notch1/2 KO OT-I cells using DESeq2 v1.30.0 R package (Love et al., 2014). DARs were annotated with their closest feature and different transcription binding motifs identified with HOMER v4.8.0. An unbiased motif search was also performed with HOMER to identify all known motifs and de novo motifs in these regions. Footprinting analysis was done with the HMM-based IdeNtification of Transcription factor footprints (HINT) library from the Regulatory Genomics Toolbox (RGT) software (version 0.13.2, (Li et al., 2019). Footprints were called from chromatin accessible regions (called by MACS2 2.6) for each sample and then matched to motifs using JASPAR 2020. Differential activity of transcription factors was assessed using HINT-differential, while p-value and read counts were calculated using RGT HINT-differential.
Assembly: mm10
Supplementary files format and content: Read counts (.xlsx), bigWig (.bw)

molecule: genomic DNA

extract_protocol:
Cells were kept on ice, washed twice in ice-cold PBS without Ca2+/Mg2+ and centrifuged for 5 minutes at 1300 rpm. Cells were resuspended in one volume of ice-cold PBS before adding four volumes of lysis buffer (12.5 mM Tris, pH 7.4; 45 mM KCl; 6.25 mM MgCl2; 375 mM sucrose; 0.125% Nonidet P-40; 1 tablet of complete [EDTA free] protease inhibitor mixture [Roche]/50 mL). After 10 minutes of incubation on ice, lysed cells were centrifuged at 4°C for 7 minutes at 500g. The nuclei pellet was resuspended in a tenth of the original lysis volume in wash buffer (10 mM Tris, pH 7.4; 60 mM KCl; 15 mM NaCl; 5 mM MgCl2; 300 mM sucrose). Then, wash buffer was added to half of the lysis volume and nuclei were centrifuged at 4°C for 7 minutes at 500g. Washed nuclei were counted, and 50,000 nuclei were used for the transposase reaction.
5 × 10^4 nuclei were directly treated with Tn5 tranposase at 37°C for 30 min. After the enzymatic reaction, the DNA was purified by Mini-Elute PCR Purification columns (Qiagen) and enriched by 12 PCR cycles. The library was recovered from PCR by GeneRead Purification columns (Qiagen) and double-strand sequenced on Illumina NovaSeq 6000 S4 PE100. A total of 112 to 132 × 10^6 reads/sample were obtained. For footprinting analysis, libraries were re-sequenced to allow increased sequencing depth, and an additional 103 to 149 × 10^6 reads/sample were obtained.


title: RNA-seq Day3_KO_1

description: Library name: Sample 15


title: Patient_2_rep7


title: LR_SN_41

description: Library name: LR_SN_41

value: brain

value: neuron

value: Naive


title: Patient_2_rep8


title: RNA-seq Day3_WT_8

description: Library name: Sample 14


title: ATAC-seq WT_2

description: Library name: Sample 2


title: LR_SN_40

description: Library name: LR_SN_40

value: female

value: DLPFC


title: RNA-seq Day3_WT_7

description: Library name: Sample 13


title: Patient_2_rep6


title: ATAC-seq WT_1

description: Library name: Sample 1


title: LR_SN_38

description: Library name: LR_SN_38


title: RNA-seq Day3_WT_5

description: Library name: Sample 11


title: Patient_2_rep5


title: LR_SN_37

description: Library name: LR_SN_37


title: ATAC-seq KO_1

description: Library name: Sample 3


title: LR_SN_39

description: Library name: LR_SN_39


title: LR_SN_36

description: Library name: LR_SN_36

value: Intox


title: RNA-seq Day3_WT_4

description: Library name: Sample 10

value: Activated OT-I CD8+ T cell


title: Patient_2_rep3


title: RNA-seq Day3_WT_6

description: Library name: Sample 12


title: Patient_2_rep4


title: LR_SN_42

description: Library name: LR_SN_42

value: Heterogeneous Stock


title: NAc_32

description: Library name: NAc_32

value: Chronic mor

value: male

value: NAc


title: NAc_30

description: Library name: NAc_30


title: RNA-seq Day3_WT_3

description: Library name: Sample 9

value: Spleen

value: WT


title: LR_SN_34

description: Library name: LR_SN_34


title: Patient_2_rep1

value: gastric mucosa

value: gastric epithelial cells


title: Patient_2_rep2


title: RNA-seq Day3_WT_2

description: Library name: Sample 8

value: Listeria monocytogenes

value: Day 3


title: NAc_29

description: Library name: NAc_29

value: brain


title: Patient_1_rep8


title: RNA-seq Day3_WT_1

description: Library name: Sample 7

value: Female


title: Patient_1_rep7


title: NAc_28

description: Library name: NAc_28

value: neuron


title: LR_SN_33

description: Library name: LR_SN_33

value: female


title: LR_SN_35

description: Library name: LR_SN_35

value: DLPFC


title: NAc_27

description: Library name: NAc_27


title: NAc_25

description: Library name: NAc_25


title: NAc_26

description: Library name: NAc_26


title: RNA-seq Naive_KO_2

description: Library name: Sample 5

source_name: Lymph nodes and spleen

value: Lymph nodes and spleen

value: Naive OT-I CD8+ T cell

value: Notch1/2 KO

value: Day 0


title: LR_SN_30

description: Library name: LR_SN_30

value: Heterogeneous Stock

value: Abstinence


title: RNA-seq Naive_KO_3

description: Library name: Sample 6


title: Patient_1_rep6


title: Patient_1_rep5


title: LR_SN_29

description: Library name: LR_SN_29


title: LR_SN_31

description: Library name: LR_SN_31

value: Intox


title: NAc_24

description: Library name: NAc_24

value: Mor + 24h

value: male

value: NAc


title: RNA-seq Naive_KO_1

description: Library name: Sample 4


title: LR_SN_32

description: Library name: LR_SN_32


title: RNA-seq Naive_WT_3

description: Library name: Sample 3

value: WT


title: NAc_23

description: Library name: NAc_23


title: Patient_1_rep4

value: gastric mucosa

value: gastric epithelial cells


title: NAc_31

description: Library name: NAc_31

value: brain

value: Chronic mor


title: tsc_bmi1_rip_rep2

city: Chapel Hill

first: Joseph

middle: Mauro

last: Calabrese

state: NC

address: 120 Mason Farm Road

institute: UNC Chapel Hill

description:
Library name: tsc_bmi1_rip_rep2
please note that processed data is linked to the rep1 sample.
tsc_bmi1_rip_-_if_reverse_stranded_F16.wig.gz
tsc_bmi1_rip_+_if_reverse_stranded_f16.wig.gz
bmi1_fc_rpm_2igg_2reps.txt

data_processing:
*library strategy: RNA immunoprecipitation sequencing (RIP-Seq)
RIP-Seq reads were aligned using STAR v2.5.4b with default parameters.
Aligned reads that had a mapping quality greater than or equal to 30 were extracted with samtools.
Filtered aligned reads were counted over features from manually curated peak files using subread featureCounts.
Assembly: mm10
Supplementary files format and content: Wiggle density files were created using a custom perl script. The WIG file includes reads pooled from the two or more biological replicates.
Supplementary files format and content: For each protein profiled by RIP, after alignment and MAPQ of ≥30 filtering, sequencing data from all replicates were concatenated split into two files using samtools, corresponding to alignments that mapped to the positive and negative strands of the genome, respectively. Using a custom perl script, the strand information within the positive and negative strand alignment files was randomized so as to better match the criteria of the MACS peak caller, which uses the average distance between positive and negative strand alignments to estimate the fragment length. Putative peaks were called on strand-randomized positive and negative strand alignment files, respectively, using default MACS parameters and not providing a background file. Peak bed files were converted to SAF format and reads under each putative peak were counted for each RIP replicate as well as for concatenated the set of MAPQ of ≥ 30 filtered reads from all IgG replicates using featureCounts. Counts per peak per dataset were converted into RPM by dividing by the total number of sequenced reads per dataset and multiplying by 1 million. We defined as peaks those regions whose RPM-normalized values in the RIPs were >2x those in IgG control in at least two RIP replicates.

source_name: Trophoblast Stem Cells

growth_protocol: TSCs were cultured as in (Quinn et al. 2006). Briefly, TSCs were cultured on gelatin-coated, pre-plated irradiated mouse embryonic fibroblast (irMEF) feeder cells in TSC media (RPMI [Gibco, cat #: 11875093], 20% qualified FBS [Gibco, cat #: 26140079], 0.1mM penicillin-streptomycin [Gibco, cat #: 15140122], 1mM sodium pyruvate [Gibco, cat #: 11360070], 2mM L-glutamine [Gibco, cat #: 25030081], 100uM beta-mercaptoethanol [Sigma-Aldrich, cat #: 63689]) supplemented with 25ng/mL FGF4 (Gibco, cat #: PHG0154) and 1ug/mL Heparin (Sigma-Aldrich, cat #: H3149) just before use, at 37°C in a humidified incubator at 5% CO2. At passage, TSCs were trypsinized with 0.125% Trypsin-EDTA in PBS solution (Gibco, cat #: 25200-072) for ~4 minutes at room temperature and gently dislodged from the plate with a sterile, cotton-plugged Pasteur pipette. To deplete irMEFs from TSCs prior to all harvests, TSCs were pre-plated for 45 minutes at 37°C, transferred to a fresh culture plate, and then cultured for three days in 70% irMEF-conditioned TSC media supplemented with growth factors as above. Mouse E14 ESCs were cultured on gelatin-coated plates in ESC media (DMEM high glucose and sodium pyruvate [Gibco, cat #: 11995073], 15% qualified FBS, 0.1mM MEM non-essential AA [Gibco, cat #: 11140050], 0.1mM penicillin-streptomycin, 2mM L-glutamine, 100M beta-mercaptoethanol, 1:500 LIF [ESC media conditioned with Lif-1Cα/COS cells]) at 37°C in a humidified incubator with 5% CO2. At passage, ESCs were trypsinized with 0.125% Trypsin-EDTA in PBS solution for ~5 minutes at room temperature and dislodged from the plate at single-cell suspension.  ESCs were passaged every other day and provided fresh media daily.

extract_protocol:
Both input and immunoprecipitated RNA were purified with the Zymo Clean and Concentrator.
KAPA RNA Hyperprep Kit with RiboErase, 9uL of IP/input RNA + 1uL ERCC RNA spike-in

treatment_protocol: Prior to crosslinking for RIP, TSCs were passaged once off of irMEFs. For all RIP experiments, trypsinized cells were crosslinked with 0.3% formaldehyde (Thermo Fisher, cat #: 28908) in PBS for 30 minutes at 4C with rotation, then quenched with 200mM glycine for 5 minutes at room temperature. Crosslinked cells were then washed thrice with ice-cold PBS, followed by resuspension in ice-cold PBS and divided into 10-million cell aliquots.

tag: antibody

value: Bmi1 (D42B3) Rabbit mAb cell signaling #5856

value: Trophoblast Stem Cells

value: wildtype


title: NAc_22

description: Library name: NAc_22

value: neuron


title: Patient_1_rep3


title: Patient_1_rep1


title: LR_SN_25

description: Library name: LR_SN_25

value: female

value: DLPFC


title: LR_SN_22

description: Library name: LR_SN_22

value: Heterogeneous Stock

value: Withdrawal


title: NAc_17

description: Library name: NAc_17


title: LR_SN_21

description: Library name: LR_SN_21


title: tsc_cytoplasm_RNA_rep2

description: Library name: tsc_cytoplasm_RNA_rep2

data_processing:
We first created a version of the GENCODE vM25 basic transcriptome that for each gene, included one representative intron-containing (i.e. nascent) transcript that began at the gene’s first annotated transcription start and the last annotated transcription end 36. We used kallisto 103 with the options “--single -s 300 -l 100 --rf-stranded” to align RNA-Seq data from total cellular RNA, as well as TSC cytoplasmic and chromatin- associated fractions.
Assembly: mm39
Supplementary files format and content: Wiggle density files were created using github.com/CalabreseLab/seekr2.0_update_manuscript/blob/main/wiggles/

molecule: cytoplasmic RNA

source_name: TSC 6-2

growth_protocol: TSCs were cultured as in (Quinn et al. 2006). Briefly, TSCs were cultured on gelatin-coated, pre-plated irradiated mouse embryonic fibroblast (irMEF) feeder cells in TSC media (RPMI [Gibco 11875093], 20% qualified FBS [Gibco 26140079], 0.1mM penicillin-streptomycin [Gibco 15140122], 1mM sodium pyruvate [Gibco 11360070], 2mM L-glutamine [Gibco 25030081], 100uM beta-mercaptoethanol [Sigma-Aldrich 63689]) supplemented with 25ng/mL FGF4 (Gibco PHG0154) and 1ug/mL Heparin (Sigma-Aldrich H3149) just before use, at 37°C in a humidified incubator at 5% CO2. At passage, TSCs were trypsinized with 0.125% Trypsin-EDTA in PBS solution (Gibco 25200-072) for ~4 minutes at room temperature and gently dislodged from the plate with a sterile, cotton-plugged Pasteur pipette. To deplete irMEFs from TSCs prior to all harvests, TSCs were pre-plated for 45 minutes at 37°C, transferred to a fresh culture plate, and then cultured for three days in 70% irMEF-conditioned TSC media supplemented with growth factors as above.

extract_protocol:
To isolate chromatin-associated, soluble nuclear, and cytoplasmic RNA, we performed a cellular fractionation as described in 65 with a few exceptions. TSCs were passaged once off irMEFs with 40 minutes of pre-plating and cultured in conditioned media on a 10cm plate. Once the 10-cm plate was 50% confluent, cells were washed twice with cold PBS and scraped in 1 mL PBS. Cells were centrifuged at 1500 x g for 5 min, and resuspended in 200 μL cold cell lysis buffer (10 mM Tris-HCl pH 7.5, 0.05% IGEPAL CA-630, 150 mM NaCl), incubating on ice for 5 min. The cell lysate was then layered over 2.5 volumes of chilled sucrose cushion (24% sucrose in cell lysis buffer) and centrifuged at 15,000 x g for 10 min at 4°C. The supernatant was removed and became the cytoplasmic fraction. The resulting pellet of nuclei was washed with PBS (pH 7.5) containing 1 mM EDTA. The pellet was then resuspended in 100 μL of cold glycerol buffer (20 mM Tris-HCl pH 7.5, 75 mM NaCl, 0.5 mM EDTA, 0.85 mM dithiothreitol [DTT], 0.125 mM 30 phenylmethylsulfonyl fluoride [PMSF], 50% glycerol) by gently flicking the tube. Then 100 μL of cold nuclei lysis buffer (10 mM HEPES pH 7.5, 1 mM DTT, 7.5 MgCl2, 0.2 mM EDTA, 0.3 M NaCl, 1 M urea, 1% IGEPAL CA-630) was added and the sample was vortexed for four seconds. After 2 min on ice, the nuclear lysate was spun at 15,000 x g for 2 min. The supernatant was removed and became the soluble nuclear (nucleoplasm) fraction. The remaining pellet (chromatin fraction) was washed with PBS containing 1 mM EDTA. To the chromatin pellet, 1 mL of TRIzol was added and vigorously resuspended by vortex and pipetting. 100 μL of cytoplasmic and nucleoplasm fractions were added to 1 mL of TRIzol and vortexed. The samples (in TRIzol) were then heated at 55°C for 10 min. The samples were then incubated on ice for 2 min. The samples were vortexed again and 200 μL CHCl3 was added. After a final vortex, samples were spun at 12,000 x g for 15 min at 4°C. The aqueous phase was extracted, and one volume of 100% ethanol was added. Samples were vortexed and RNA was extracted using Zymo-Spin IC Columns (from Zymo #R1013). RNA was eluted in 50 μL of RNase-free water. RNA concentrations were measured via Qubit 2.0 fluorometer (RNA high sensitivity kit, Thermo Fisher Q32852), and integrity of RNA was confirmed by visualizing rRNA bands  on an agarose gel, also confirming an absence of rRNA in the nucleoplasm and chromatin fractions. RNA samples were stored at -80°C.
Libraries for RNA sequencing were prepared using the KAPA RNA HyperPrep Kit with RiboErase (HMR) (Roche 08098140702) and KAPA Unique Dual-Indexed Adapters (Roche 08861919702) or TruSeq adapters, with each library’s input consisting of 9μL 100ng/μL total RNA (900 ng) mixed with 1μL of a 1:250 dilution of ERCC RNA spike-in controls (Thermo Fisher 4456740). RNA fragmentation was performed at 94°C for 6min. Libraries were pooled and sequenced on an Illumina NextSeq500 platform with a 75-cycle NextSeq 500/550 High Output Kit v2.5 (Illumina 20024906) or  on an Illumina NextSeq1000 platform with a 100-cycle NextSeq 1000/2000 P2 Reagents v3 kit (Illumina 20046811).

value: TSC 6-2

value: Cellular Fractionation


title: RNA-seq Naive_WT_1

description: Library name: Sample 1

value: Lymph nodes and spleen

value: Naive OT-I CD8+ T cell

value: Day 0

value: Female


title: LR_SN_28

description: Library name: LR_SN_28

value: Abstinence


title: Patient_1_rep2

value: gastric mucosa

value: gastric epithelial cells


title: LR_SN_26

description: Library name: LR_SN_26

value: brain


title: NAc_19

description: Library name: NAc_19

value: Mor + 24h

value: NAc


title: NAc_20

description: Library name: NAc_20

value: neuron


title: LR_SN_23

description: Library name: LR_SN_23

value: Intox

value: male


title: tsc_bmi1_rip_rep1

description:
Library name: tsc_bmi1_rip_rep1
tsc_bmi1_rip_-_if_reverse_stranded_F16.wig.gz
tsc_bmi1_rip_+_if_reverse_stranded_f16.wig.gz
bmi1_fc_rpm_2igg_2reps.txt

value: Bmi1 (D42B3) Rabbit mAb cell signaling #5856

value: Trophoblast Stem Cells

value: wildtype


title: NAc_21

description: Library name: NAc_21

value: female


title: LR_SN_27

description: Library name: LR_SN_27

value: Heterogeneous Stock

value: DLPFC


title: NAc_18

description: Library name: NAc_18


title: tsc_cytoplasm_RNA_rep1

description: Library name: tsc_cytoplasm_RNA_rep1

value: TSC 6-2

value: Cellular Fractionation


title: LR_SN_24

description: Library name: LR_SN_24


title: RNA-seq Naive_WT_2

description: Library name: Sample 2

value: Lymph nodes and spleen

value: Naive OT-I CD8+ T cell

value: WT

value: Day 0

value: Female


title: tsc_nuclear_RNA_rep1

description: Library name: tsc_nuclear_RNA_rep1

molecule: nuclear RNA


title: NAc_16

description: Library name: NAc_16

value: brain

value: Mor + 24h

value: NAc


title: tsc_chromatin_RNA_rep1

description: Library name: tsc_chromatin_RNA_rep1


title: tsc_chromatin_RNA_rep2

description: Library name: tsc_chromatin_RNA_rep2


title: NAc_15

description: Library name: NAc_15

value: neuron

value: Mor + 2W

value: male


title: LR_SN_20

description: Library name: LR_SN_20

value: Naive


title: osteochondral fusion

city: Chengdu

first: Zhonghan

last: Li

state: Sichuan

address: 29 Wangjiang Road

department: College of Life Sciences

country: China

institute: Sichuan University

data_processing:
Sequencing data from 10x Genomics were processed using CellRanger (ver. 7.1.0) for demultiplexing, barcode processing, and single-cell 3’-gene counting. Human (GRCh38) and mouse (GRCm39) reference-20240A was used for sequence alignment.
Assembly: Human (GRCh38) and mouse (GRCm39) reference-20240A
Supplementary files format and content: Tab-seperated values files and matrix files

source_name: xenografts carried in NOD/SCID mice generated from hPSC-derived cells

extract_protocol:
To collect enough cells for single-cell RNA sequencing, three osteochondral fusions were pooled together and were dissociated to single cells by Liberase after cut into small fragments. Digestion was stopped until no large fragments could be found and the cells were filtered by 70μm strainers. After that, the cells were treated with hemolysis reagents for 10 min at RT. Then 1 mL basal medium composed of DMEM/F12, 10% FBS, 1% pen-strep was added and the cells were centrifuged at 300g for 5 min and were resuspended in the medium mentioned above for subsequent filtering with 40μm strainers
Library was constructed under manufacture's instruction (Chromium Single Cell 3’ Library & Single Cell 3ʹ v3 Gel Beads)

value: cartilage/bone

tag: cell line

value: bone/cartilage tissues generated from hPSC-derived osteochondral fusion grafts

value: hPSC-derived osteochondral fusions that were transplanted under kidney capsule of the NOD/SCID mice


title: NAc_14

description: Library name: NAc_14


title: Untreated X872-Airn-RMCE clone 2, rep 2 RNA

description:
Library name: XA2_no_dox_rep2_RNA_S7_R1_001.fastq.gz
wiggle is pooled replicates and downsampled and merged
X872A_no_dox_RNA_+_if_reverse_stranded_f16.wig.gz
X872A_no_dox_RNA_-_if_reverse_stranded_F16.wig.gz

data_processing:
Sequencing reads (fastq files) were mapped both to the GENCODE vM25 mouse genome (“basic CHR” from https://www.gencodegenes.org/mouse/release_M25.html) [for B6] and to a version of the same genome modified to incorporate CAST single-nucleotide polymorphisms (SNPs) [for CAST]. Mapping was performed with STAR (v2.7.9a), using two-pass mapping and the option “--outFilterMultimapNmax 1” to consider only uniquely mapping reads.
Using a custom Perl script (intersect_reads_snps16.pl) and a file containing a list of CAST SNPs (sangercast07_30_20), aligned reads in the B6 and CAST SAM files were parsed to identify reads clearly originating from either the B6 or CAST allele (i.e. reads that overlap at least 1 B6 or CAST SNP).
For genome browser viewing, strand-specific mm10-aligned reads were separated using samtools: samtools view -Sq 30 (negative) -F 0x10 or (positive) -f 0x10
Assembly: mm10
Supplementary files format and content: Wiggle density files were created using github.com/CalabreseLab/seekr2.0_update_manuscript/blob/main/wiggles/

source_name: RMCE Embryonic Stem Cells

growth_protocol: B6/CAST F1-hybrid Rosa26-RMCE ESCs were grown on gelatin-coated plastic dishes in a humidified Thermo Fisher Forma Series II water-jacketed incubator at 37°C and under 5% CO2. Cells were grown in DMEM high glucose plus sodium pyruvate (Gibco 11995-065), 15% ESC-qualified fetal bovine serum (Gibco 26140-079), 0.1mM non-essential amino acids (Gibco 11140-050), 100U/mL penicillin-streptomycin (Gibco 15140-122), 2mM L-glutamine (Gibco 25030-081), 0.1mM β-mercaptoethanol, and 1:500 LIF conditioned media produced from Lif-1Cα (COS) cells (kind gift of N. Hathaway). TSCs were cultured as in (Quinn et al. 2006). Briefly, TSCs were cultured on gelatin-coated, pre-plated irradiated mouse embryonic fibroblast (irMEF) feeder cells in TSC media (RPMI [Gibco 11875093], 20% qualified FBS [Gibco 26140079], 0.1mM penicillin-streptomycin [Gibco 15140122], 1mM sodium pyruvate [Gibco 11360070], 2mM L-glutamine [Gibco 25030081], 100uM beta-mercaptoethanol [Sigma-Aldrich 63689]) supplemented with 25ng/mL FGF4 (Gibco PHG0154) and 1ug/mL Heparin (Sigma-Aldrich H3149) just before use, at 37°C in a humidified incubator at 5% CO2. At passage, TSCs were trypsinized with 0.125% Trypsin-EDTA in PBS solution (Gibco 25200-072) for ~4 minutes at room temperature and gently dislodged from the plate with a sterile, cotton-plugged Pasteur pipette. To deplete irMEFs from TSCs prior to all harvests, TSCs were pre-plated for 45 minutes at 37°C, transferred to a fresh culture plate, and then cultured for three days in 70% irMEF-conditioned TSC media supplemented with growth factors as above.

extract_protocol:
RMCE ESCs or MEF feeder-free TSCs were washed twice with ice-cold PBS and harvested with 1 mL TRIzol (Thermo Fisher 15596026). Total RNA was prepared via standard TRIzol-chlorofom extraction, per manufacturer’s instructions, except for the addition of 4μL 5μg/μL linear acrylamide (Thermo Fisher AM9520) to promote precipitation in isopropanol. RNA pellets were fully resuspended in RNase-free water, RNA concentrations were measured via Qubit 2.0 fluorometer (RNA high sensitivity kit, Thermo Fisher Q32852), and integrity of RNA was confirmed by visualizing rRNA bands on an agarose gel. Working stocks of 100ng/μL total RNA were prepared, and all RNA samples were stored at -80°C.
Libraries for RNA sequencing were prepared using the KAPA RNA HyperPrep Kit with RiboErase (HMR) (Roche 08098140702) and KAPA Unique Dual-Indexed Adapters (Roche 08861919702) or TruSeq adapters, with each library’s input consisting of 9μL 100ng/μL total RNA (900 ng) mixed with 1μL of a 1:250 dilution of ERCC RNA spike-in controls (Thermo Fisher 4456740). RNA fragmentation was performed at 94°C for 6min. Libraries were pooled and sequenced on an Illumina NextSeq500 platform with a 75-cycle NextSeq 500/550 High Output Kit v2.5 (Illumina 20024906) or  on an Illumina NextSeq1000 platform with a 100-cycle NextSeq 1000/2000 P2 Reagents v3 kit (Illumina 20046811).

treatment_protocol: Prior to harvesting, RMCE cell lines (with rtTA) were treated with 0-1000ng/ml doxycycline (1000ng/ml if not otherwise specified) for 72 hours (or longer, 7 days).

value: RMCE Embryonic Stem Cells


title: NAc_12

description: Library name: NAc_12


title: NAc_13

description: Library name: NAc_13


title: 3-day 1000ng/mL dox-treated X872-Airn-RMCE clone 2 RNA rep 2

description:
Library name: XA2_dox_rep2_RNA_S8_R1_001.fastq.gz
wiggle is pooled replicates and downsampled and merged
X872A_dox_RNA_+_if_reverse_stranded_f16.wig.gz
X872A_dox_RNA_-_if_reverse_stranded_F16.wig.gz

value: 3-day 1000ng/mL dox-treated


title: 3-day 1000ng/mL dox-treated X872-Airn-RMCE clone 2 RNA rep 1

description:
Library name: XA2_dox_rep1_RNA_S6_R1_001.fastq.gz
wiggle is pooled replicates and downsampled and merged
X872A_dox_RNA_+_if_reverse_stranded_f16.wig.gz
X872A_dox_RNA_-_if_reverse_stranded_F16.wig.gz


title: LR_SN_17

description: Library name: LR_SN_17

value: Heterogeneous Stock

value: DLPFC


title: NAc_11

description: Library name: NAc_11


title: LR_SN_19

description: Library name: LR_SN_19


title: LR_SN_18

description: Library name: LR_SN_18


title: Untreated X872-Airn-RMCE clone 1, rep 1 RNA

description:
Library name: XA1_no_dox_rep1_RNA_S1_R1_001.fastq.gz
wiggle is pooled replicates and downsampled and merged
X872A_no_dox_RNA_+_if_reverse_stranded_f16.wig.gz
X872A_no_dox_RNA_-_if_reverse_stranded_F16.wig.gz


title: NAc_9

description: Library name: NAc_9

value: female


title: rmce_Airn5_igg_rip

description:
Library name: rmce_Airn5_igg_rip
RIP
Airn_igg_-_if_reverse_stranded_F16.wig.gz
Airn_igg_+_if_reverse_stranded_f16.wig.gz

library_source: other

data_processing:
RIP-Seq reads were aligned using STAR v2.5.4b with default parameters.
Aligned reads that had a mapping quality greater than or equal to 30 were extracted with samtools.
Assembly: mm10
Supplementary files format and content: Wiggle density files were created using github.com/CalabreseLab/seekr2.0_update_manuscript/blob/main/wiggles/

molecule: other

growth_protocol: Mouse E14 ESCs were cultured on gelatin-coated plates in ESC media (DMEM high glucose and sodium pyruvate [Gibco, cat #: 11995073], 15% qualified FBS, 0.1mM MEM non-essential AA [Gibco, cat #: 11140050], 0.1mM penicillin-streptomycin, 2mM L-glutamine, 100M beta-mercaptoethanol, 1:500 LIF [ESC media conditioned with Lif-1Cα/COS cells]) at 37°C in a humidified incubator with 5% CO2. At passage, ESCs were trypsinized with 0.125% Trypsin-EDTA in PBS solution for ~5 minutes at room temperature and dislodged from the plate at single-cell suspension.  ESCs were passaged every other day and provided fresh media daily.

treatment_protocol: For all RIP experiments, trypsinized cells were crosslinked with 0.3% formaldehyde (Thermo Fisher, cat #: 28908) in PBS for 30 minutes at 4C with rotation, then quenched with 200mM glycine for 5 minutes at room temperature. Crosslinked cells were then washed thrice with ice-cold PBS, followed by resuspension in ice-cold PBS and divided into 10-million cell aliquots.

value: 10ug Invitrogen #026102

value: Airn-RMCE clone 5


title: NAc_10

description: Library name: NAc_10

value: brain


title: 3-day 1000ng/mL dox-treated X872-Airn-RMCE clone 1 RNA rep 1

description:
Library name: XA1_dox_rep1_RNA_S2_R1_001.fastq.gz
wiggle is pooled replicates and downsampled and merged
X872A_dox_RNA_+_if_reverse_stranded_f16.wig.gz
X872A_dox_RNA_-_if_reverse_stranded_F16.wig.gz


title: NAc_6

description: Library name: NAc_6

value: Sal


title: LR_SN_13

description: Library name: LR_SN_13

value: Abstinence


title: NAc_7

description: Library name: NAc_7

value: neuron

value: NAc


title: 3-day 1000ng/mL dox-treated X872-Airn-RMCE clone 1 RNA rep 2

description:
Library name: XA1_dox_rep2_RNA_S4_R1_001.fastq.gz
wiggle is pooled replicates and downsampled and merged
X872A_dox_RNA_+_if_reverse_stranded_f16.wig.gz
X872A_dox_RNA_-_if_reverse_stranded_F16.wig.gz


title: LR_SN_10

description: Library name: LR_SN_10

value: male


title: rmce_Airn2_igg_rip

description:
Library name: rmce_Airn2_igg_rip
RIP
Airn_igg_-_if_reverse_stranded_F16.wig.gz
Airn_igg_+_if_reverse_stranded_f16.wig.gz

value: Airn-RMCE clone 2


title: NAc_8

description: Library name: NAc_8

value: Mor + 2W


title: LR_SN_12

description: Library name: LR_SN_12


title: LR_SN_14

description: Library name: LR_SN_14


title: NAc_5

description: Library name: NAc_5


title: LR_SN_11

description: Library name: LR_SN_11


title: Untreated X872-Airn-RMCE clone 2, rep 1 RNA

description:
Library name: XA2_no_dox_rep1_RNA_S5_R1_001.fastq.gz
wiggle is pooled replicates and downsampled and merged
X872A_no_dox_RNA_+_if_reverse_stranded_f16.wig.gz
X872A_no_dox_RNA_-_if_reverse_stranded_F16.wig.gz


title: LR_SN_16

description: Library name: LR_SN_16

value: Naive


title: NAc_4

description: Library name: NAc_4


title: Untreated X872-Airn-RMCE clone 1, rep 2 RNA

description:
Library name: XA1_no_dox_rep2_RNA_S3_R1_001.fastq.gz
wiggle is pooled replicates and downsampled and merged
X872A_no_dox_RNA_+_if_reverse_stranded_f16.wig.gz
X872A_no_dox_RNA_-_if_reverse_stranded_F16.wig.gz


title: LR_SN_15

description: Library name: LR_SN_15


title: LR_SN_9

description: Library name: LR_SN_9


title: NAc_3

description: Library name: NAc_3


title: rmce_XA2_igg_rip

description:
Library name: rmce_XA2_igg_rip
RIP
X872A_igg_-_if_reverse_stranded_F16.wig.gz
X872A_igg_+_if_reverse_stranded_f16.wig.gz

value: X872-Airn-RMCE clone 2


title: rmce_XA1_igg_rip

description:
Library name: rmce_XA1_igg_rip
RIP
X872A_igg_-_if_reverse_stranded_F16.wig.gz
X872A_igg_+_if_reverse_stranded_f16.wig.gz

value: RMCE Embryonic Stem Cells

value: X872-Airn-RMCE clone 1


title: NAc_2

description: Library name: NAc_2


title: LR_SN_8

description: Library name: LR_SN_8

value: Heterogeneous Stock

value: Withdrawal


title: rmce_Airn5_spen_rip

description:
Library name: rmce_Airn5_spen_rip
RIP
Airn_spen_-_if_reverse_stranded_F16.wig.gz
Airn_spen_+_if_reverse_stranded_f16.wig.gz

value: SPEN Antibody NovusNBP1-82952

value: 3-day 1000ng/mL dox-treated


title: LR_SN_7

description: Library name: LR_SN_7

value: DLPFC


title: rmce_Airn2_spen_rip

description:
Library name: rmce_Airn2_spen_rip
RIP
Airn_spen_-_if_reverse_stranded_F16.wig.gz
Airn_spen_+_if_reverse_stranded_f16.wig.gz


title: LR_SN_5

description: Library name: LR_SN_5

value: brain


title: PFC_31

description: Library name: PFC_31

value: Chronic mor

value: PFC


title: rmce_XA2_spen_rip

description:
Library name: rmce_XA2_spen_rip
RIP
X872A_spen_-_if_reverse_stranded_F16.wig.gz
X872A_spen_+_if_reverse_stranded_f16.wig.gz


title: NAc_1

description: Library name: NAc_1

value: neuron

value: Sal

value: female


title: PFC_30

description: Library name: PFC_30

value: male


title: rmce_XA1_spen_rip

description:
Library name: rmce_XA1_spen_rip
RIP
X872A_spen_-_if_reverse_stranded_F16.wig.gz
X872A_spen_+_if_reverse_stranded_f16.wig.gz

value: SPEN Antibody Novus NBP1-82952


title: LR_SN_2

description: Library name: LR_SN_2

value: Intox


title: 3-day 1000ng/mL dox-treated Xist-RMCE ESC clone 1 H2AK119ub

data_processing:
ChIP-Seq reads were aligned using bowtie2 with default parameters.
Aligned reads that had a mapping quality greater than or equal to 30 were extracted with samtools 28, and allele-specific read retention (i.e., reads that overlap at least one B6 or CAST SNP) was performed as in 30,31 using a custom Perl script (intersect_reads_snps16.pl).
Assembly: mm10
Supplementary files format and content: WIG files were created using a custom perl script. Total, non-allelic read counts are divided by total reads of the dataset in million (i.e. RPM).

growth_protocol: B6/CAST F1-hybrid Rosa26-RMCE ESCs from 12 were grown on gelatin-coated plastic dishes in a humidified Thermo Fisher Forma Series II water-jacketed incubator at 37°C and under 5% CO2. Cells were grown in DMEM high glucose plus sodium pyruvate (Gibco 11995-065), 15% ESC-qualified fetal bovine serum (Gibco 26140-079), 0.1mM non-essential amino acids (Gibco 11140-050), 100U/mL penicillin-streptomycin (Gibco 15140-122), 2mM L-glutamine (Gibco 25030-081), 0.1mM β-mercaptoethanol (Sigma-Aldrich 63689), and 1:500 LIF conditioned media produced from Lif-1Cα (COS) cells (kind gift of N. Hathaway).

extract_protocol:
ChIP beads were resuspended in Elution buffer (50mM Tris-HCl pH 8.0, 10mM EDTA, 1% SDS) and placed on a 65°C heat block for 17 minutes with frequent vortexing. ChIP DNA was then reverse crosslinked in 0.5% SDS and 100mM NaCl overnight at 65°C, followed by a 1-h RNaseA (3L; Thermo Scientific EN0531) treatment at 37°C and a 2.5-h Proteinase K (10L; Invitrogen 25530015) treatment at 56°C. DNA was then extracted with 1 volume of phenol:chloroform:isoamyl alcohol (Sigma-Aldrich P3803) and precipitated with 2 volumes 100% ethanol, 1/10 volume 3M sodium-acetate pH 5.4, and 1/1000 volume linear acrylamide (Invitrogen AM9520) overnight at -20°C. Precipitated DNA was then extracted with a 30-minute max speed spin at 4°C, washed once with ice-cold 80% ethanol, and resuspended in TE.
ChIP-Seq libraries were prepared with NEBNext End Repair Module (NEB, cat #: E6050S), A-tailing by Klenow Fragment (3’--5’ exo-; NEB, cat #: M0212S), and TruSeq 6-bp index adaptor ligation by Quick ligase (NEB, cat #: M2200S), and NEBNext High-Fidelity 2X PCR Master Mix (NEB, cat #: M0541S). All DNA clean-up steps were performed with AMPure XP beads (Beckman Coulter, cat #: A63880). Single-end, 75-bp sequencing was performed using an Illumina NextSeq 500/550 High Output v2.5 kit (Illumina, cat #: 20024906) on a NextSeq 500 System.

treatment_protocol: Prior to crosslinking, RMCE cell lines (with rtTA) were treated with 0-1000ng/mL doxycycline (1000ng/mL if not otherwise specified) for 72 hours (or longer, 7 days). Adhered cells were crosslinked with 0.6% formaldehyde (Fisher Scientific BP531-500) in DMEM media with 5% FBS for 10min at room temperature, then quenched with 125mM glycine for 5min at room temperature. Crosslinked cells were then washed twice with ice-cold PBS and scraped with ice-cold PBS with 0.05% Tween (Fisher Scientific EW-88065-31) and PIC (Sigma Aldrich P8340). The cells were then spun at 1,200 x g at 4°C to remove PBS, followed by resuspension in ice-cold PBS with PIC and divided into 10-million cell aliquots.

value: 3 days with 1000ng/mL doxycycline

value: Xist-RMCEs

tag: chip antibody

value: Ubiquityl-Histone H2A (Lys119) (D27C4) (H2AK119ub) (Cell Signaling, cat #:8240)


title: 3-day 1000ng/mL dox-treated Xist-RMCE ESC clone 4 H2AK119ub

value: RMCE Embryonic Stem Cells


title: PFC_28

description: Library name: PFC_28


title: LR_SN_4

description: Library name: LR_SN_4

value: Heterogeneous Stock


title: PFC_32

description: Library name: PFC_32


title: 3-day 10ng/mL dox-treated Xist-RMCE ESC clone 4 H2AK119ub

value: 3 days with 10ng/mL doxycycline


title: PFC_29

description: Library name: PFC_29


title: PFC_27

description: Library name: PFC_27


title: LR_SN_1

description: Library name: LR_SN_1


title: C elegans, Glia, Male, Rep2

city: Seattle

first: Manu

last: Setty

state: WA

address: 1100 Fairview Ave N

department: Basic Sciences Division

institute: Fred Hutchinson Cancer Center

description:
10X Genomics
Day1_Male_mir228GFP_Him5_Rep2

data_processing:
Count matrices for the two samples were generated using CellRanger-5.0.1
The 3’ untranslated regions (UTRs) in the reference transcriptome (WormBase WS280) have been previously reported to be too short for reliable gene assignment. Therefore, following the previously published approaches, we extended 3’ UTRs in the WS280 genome. Specifically, for genes not overlapping with a downstream gene, we extended their 3’ UTRs by 50, 100, 150, 200, 250, 300, 400 and 500bp. In case the extended sequence collided with a downstream gene, it was trimmed back to the base next to the gene. The unmodified and eight modified GTF files were each used to build a reference genome. Protein-coding genes and non-coding RNA genes (lincRNAs, ncRNAs and antisense RNAs) were included for gene expression count determination. Cellranger was used to generate a count matrix for each sample relative to each reference and the optimal 3’ UTR extension was defined as follows: 20 reads were considered as a significant gain by an extension interval. For each gene, the cumulative sums from 3’ (the longest extension) to 5’ was calculated after subtracting 20 from read increment in each extension interval. The optimal extension for a given sample was set to the point which had the smallest cumulative sum of less than 0. If cumulative sums were all greater than zero, the optimal extension was set to 500bp. The final extension of a gene was set to the point supported by most samples and by at least two samples. Prior work has suggested low UMIs for C. elegans single-cell RNAseq data59. We thus evaluated multiple cut-offs and empirically selected 200 UMIs as empty droplet cut-off. Cell-containing droplets by 10X Cellranger were used for downstream analysis
Data processing was performed using the scanpy package with default parameters unless specified. Count matrices from all samples were concatenated. Raw counts were normalized by dividing the counts by total counts per cell. The normalized data was multiplied by the median of total counts across cells to avoid numerical issues and log-transformed with a pseudo count of 1. Feature selection was then performed to select the top 2500 most highly variable genes (using scanpy.pp.highly_variable_genes), which was used as input for principal component analysis with 50 components. The PCs were used as input for generating UMAPs (with min_dist=0.2) and clustering with Leiden.
Assembly: WormBase WS280
Supplementary files format and content: Scanpy anndata objects containing counts, normalized data and cell-types

organism: Caenorhabditis elegans

source_name: 1 day old whole male worm

growth_protocol: Preparation of adults for dissociation: Worms were grown on 10 cm, standard 1% NGM agar plates seeded with E.coli strain OP50. To obtain synchronized day 1 adult worms, we first obtained embryos by performing hypochlorite treatment (1M NaOH + 4% NaOCl freshly made) on adult hermaphrodites. The embryos hatched overnight (16-20 hours) on unseeded NGM agar plates and starved L1 arrested worms were recovered on 10 cm OP50-seeded plates at 20C for 64-68 hours. For each hermaphrodite sample, about 50,000 day 1 adult worms were washed off plates with M9 into 50 ml conical tubes that were fitted with a 50um pluriStrainer® filter to separate day 1 adults from embryos or hatched L1s. The pure day 1 adult hermaphrodite population was transferred to 15 ml conical tubes and further washed 3x with M9 with centrifugation at 1200 rcf for 1 min.  For each male sample, about 150,000 day 1 adult worms were washed off plates with M9 into 50 ml conical tubes that were fitted with stacked 50 um, 40 um, and 30 um pluriStrainer® filters to separate adult males from hermaphrodites. The stacked filtration was repeated one more time. The worms from the 30 um filter were mix of about 80-90% males, plus young day 1 adult hermaphrodites. To further enrich for a male only population, we used the COPAS worm sorter to select animals based on size and lack of Pmig-24: mVenus staining and recovered 50,000 males. After sorting, 5x10ul aliquots of worms were visualized under a light microscope for counting and each aliquot contained 100% males. The males were further washed 3x with M9 with centrifugation at 1200 rcf for 1 min.

extract_protocol:
Dissociation: Single nuclei suspensions were obtained as described in Kalestky R et al., 2018 for cells, with some modifications to further dissociate the cell membranes. After removing most of the M9 solution, worms were quickly washed once in 500ul lysis buffer (200mM DTT, 0.25% SDS, 20mM Hepes pH 8.0, 3% sucrose), and then lysed with 750ul lysis buffer for 5 min for hermaphrodites and 3 minutes for males. Lysis was shorter for males because longer lysis time caused the males to rupture during the downstream M9 washing steps. Cuticle disruption was monitored by examining 1μL of sample under a microscope. When lysis was complete, worms had a slightly blunt head, but were not ruptured. Reaction was stopped by adding 500ul M9 and then did 6 quick washed in M9. Worms were then dissociated by adding 500 ul freshly made 20mg/ml Pronase (resuspended in water) to the worm pellet for 15-20 minutes depending on the lot of Pronase. Males were dissociated in 15 mg/ml Pronase. During the Pronase incubation, worms were dissociated by pipetting vigorously with a P1000 pipette set to 400ul every 45 seconds, rotating between samples for the first 10 minutes, followed by trituration with a P200 pipette set to 200 ul for remaining 5-10 minutes. Worms were quickly spun down between triturations so that the worm pellet size could be monitored. Between pipetting, 2ul of worms were examined on a dissecting scope to assess level of dissociation. Dissociation was continued until large fragments were gone and the pellet of worms was less than 20 ul when quickly centrifuged. We noticed that during this over digestion step, the cell membranes would also digest. To stop the digestion, 750μL of ice-cold 1xPBS/10%FBS was added to the tube. The dissociated nuclei were transferred to a 1ml syringe using a 27-gauge needle. The needle was replaced by a 5uM syringe filter that was pre-wetted with ice cold PBS. The cells were gently passed through the syringe filter directly into the FACS collection tube on ice. Protector RNAse Inhibitor was added to the following solutions: Pronase, PBS-FBS, and PBS-L15-FBS to get final concentration of 2U/ul inhibitor.   Cell sorting and preparation for RNA sequencing: The nuclei were sorted with a Sony SH800S cell sorter fitted with the 100µm nozzle running at a speed of 10-12k events per second using the PE filter set. Nuclei were sorted into 1:1 1xPBS:L15+30%FBS 2U/µl Protector RNAse inhibitor, transferred to a low bind 2 ml tube and centrifuged 9600xg for 5 min at 4C. Once an RFP+ pellet was confirmed under a fluorescent dissection scope, the nuclei were gently resuspended in 1xPBS10%FBS buffer aiming for a final concentration of 1000 nuclei/uL based on the number of nuclei sorted.
We targeted 10,000 cells per sample and used the 10X Chromium platform using 10X NextGem v3.1 chemistry. cDNA was amplified for 12 cycles and the index PCR was cycled 12 times. Final libraries were brought to 10 nM concentration.
Next-generation sequencing libraries were constructed following the User Guide, which were sequenced on an Illumina NovaSeq 2500 system.

value: 1 day old whole male worm

value: glia

value: miR228-GFP, Him5


title: PFC_26

description: Library name: PFC_26

value: brain

value: Chronic mor


title: PFC_25

description: Library name: PFC_25

value: neuron

value: PFC


title: Untreated Xist-RMCE ESC clone 4 H2AK119ub

value: Untreated


title: LR_SN_6

description: Library name: LR_SN_6

value: Withdrawal

value: male

value: DLPFC


title: Untreated Xist-RMCE ESC clone 1 H2AK119ub


title: PFC_24

description: Library name: PFC_24

value: Mor + 24h


title: C elegans, Glia, Male, Rep1

description:
10X Genomics
Day1_Male_mir228GFP_Him5_Rep1


title: 3-day 1000ng/mL dox-treated Airn-RMCE ESC clone 5 H2AK119ub

value: 3 days with 1000ng/mL doxycycline

value: Airn-RMCEs


title: LR_SN_3

description: Library name: LR_SN_3

value: Intox


title: PFC_22

description: Library name: PFC_22


title: 3-day 1000ng/mL dox-treated Airn-RMCE ESC clone 2 H2AK119ub

value: RMCE Embryonic Stem Cells

value: Ubiquityl-Histone H2A (Lys119) (D27C4) (H2AK119ub) (Cell Signaling, cat #:8240)


title: PFC_23

description: Library name: PFC_23


title: PFC_21

description: Library name: PFC_21

value: female


title: Input, NIPBLmut, rep1

city: Bunkyo-ku

first: Katsuhiko

last: Shirahige

state: Tokyo

address: 1-1-1 Yayoi

department: Institute for Quantitative Biosciences

country: Japan

institute: The University of Tokyo

data_processing:
Sequenced reads were aligned to the human genome build hg38 and mouse genome build mm10 using Bowtie version 1.2.2 with the options “-n2 -m1” and the uniquely mapped reads were used for subsequent analysis.
BigWig files (100-bp bin) were generated by DROMPA version 3.7.2. The read distribution is normalized based on the number of reads derived from the mouse spike-in.
Assembly: UCSC hg38
Supplementary files format and content: BigWig files (100-bp bin) of the normalized read distribution.

growth_protocol: 293FT cells were maintained in DMEM supplemented with penicilline-streptomycin-L-glutamine solution (Wako) and 10% fetal bovine serum (FBS) in humidified atmosphere with 5% CO2 at 37C.

extract_protocol:
Chromatin immunoprecipitation was performed as detailed in Nakato et al. (Nature Communications (2023)14:5647). Briefly, human culture cells were crosslinked with 1 % formaldehyde for 10 minutes at room temperature. Chromatin was then sheared by sonication (Branson Sonifier 250D) and combined with mouse spike-in chromatin. The immunoprecipitation was performed overnight at 4°C with an antibody conjugated to Dynabeads Protein A or B (Thermo Fisher Scientific, 10002D or 10004D). After completing the immunoprecipitation and reversing crosslinks, the DNA was purified.
Sequencing library was prepared for sequencing using the NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolabs, E7645) according to manufacturer's instructions.

value: NIPBLmut

tag: target protein

value: Input


title: Untreated Airn-RMCE ESC clone 5 H2AK119ub


title: Input, NIPBLwt, rep1

value: NIPBLwt


title: H3K27ac ChIP, NIPBLmut

value: anti-H3K27ac (MABI0309, MBL International)


title: H3K27ac ChIP, NIPBLwt


title: 3-day 1000ng/mL dox-treated Empty-RMCE ESC clone 4 H2AK119ub

value: Empty-RMCEs


title: Untreated Empty-RMCE ESC clone 4 H2AK119ub


title: Untreated Airn-RMCE ESC clone 2 H2AK119ub


title: BRD4 ChIP, NIPBLmut

value: anti-BRD4 (A301-985A50, BETHYL)


title: PFC_19

description: Library name: PFC_19

value: brain

value: neuron

value: PFC


title: PFC_20

description: Library name: PFC_20


title: PFC_18

description: Library name: PFC_18


title: 3-day 10ng/mL dox-treated Xist-RMCE ESC clone 1 H2AK119ub

value: 3 days with 10ng/mL doxycycline

value: Xist-RMCEs


title: tsc_rip_input_rep9

description:
RIP
input_ns.wig.gz
input_ps.wig.gz
input_fc_rpm_2igg_2reps.txt

data_processing:
RIP-Seq reads were aligned using STAR v2.5.4b with default parameters.
Aligned reads that had a mapping quality greater than or equal to 30 were extracted with samtools.
Filtered aligned reads were counted over features from manually curated peak files using subread featureCounts.
Assembly: mm10
Supplementary files format and content: Wiggle density files were created using a custom perl script. The WIG file includes reads pooled from the two or more biological replicates (ns.wig.gz represents negative strand, ps.wig.gz represents positive strand).
Supplementary files format and content: For each protein profiled by RIP, after alignment and MAPQ of >30 filtering, sequencing data from all replicates were concatenated split into two files using samtools, corresponding to alignments that mapped to the positive and negative strands of the genome, respectively. Using a custom perl script, the strand information within the positive and negative strand alignment files was randomized so as to better match the criteria of the MACS peak caller, which uses the average distance between positive and negative strand alignments to estimate the fragment length. Putative peaks were called on strand-randomized positive and negative strand alignment files, respectively, using default MACS parameters and not providing a background file. Peak bed files were converted to SAF format and reads under each putative peak were counted for each RIP replicate as well as for concatenated the set of MAPQ of >30 filtered reads from all IgG replicates using featureCounts. Counts per peak per dataset were converted into RPM by dividing by the total number of sequenced reads per dataset and multiplying by 1 million. We defined as peaks those regions whose RPM-normalized values in the RIPs were >2x those in IgG control in at least two RIP replicates.

source_name: Trophoblast stem cells

extract_protocol:
Ribo-depleted RNA
Both input and immunoprecipitated RNA were purified with the Zymo Clean and Concentrator.
KAPA RNA Hyperprep Kit with RiboErase, 9uL of IP/input RNA + 1uL ERCC RNA spike-in

tag: rip antibody

value: Trophoblast stem cells

tag: passage number

value: Wildtype


title: PFC_17

description: Library name: PFC_17

tag: tissue

value: Mor + 24h

value: male


title: BRD4 ChIP, NIPBLwt


title: tsc_rip_input_rep8


title: PFC_16

description: Library name: PFC_16

tag: cell type


title: NIPBL ChIP, NIPBLmut

value: NIPBL

value: anti-NIPBL (A301-779A, BETHYL)


title: NIPBL ChIP, NIPBLwt


title: tsc_rip_input_rep7


title: PFC_15

description: Library name: PFC_15

tag: strain

value: Mor + 2W


title: RAD21 ChIP, NIPBLmut

value: NIPBLmut

value: anti-RAD21 (in house)


title: tsc_rip_input_rep6

tag: treatment


title: tsc_rip_input_rep4


title: PFC_13

description: Library name: PFC_13

tag: batch


title: NIPBLmut, rep2

data_processing:
BAM-formatted map files were generated using STAR version 2.7.3a with default parameters.
Assembly: UCSC hg38
Supplementary files format and content: The expression values were estimated by RSEM v1.3.1 with the option "--estimate-rspd --strandedness reverse".

extract_protocol:
Total RNA was isolated using Trizol (Thermo Fisher Scientific) and Nucleospin RNA kit (Macherey-Nagel) following the manufacturer's instructions. Removal of rRNA was performed by using Ribo-Zero Gold rRNA Removal Kit (Illumina).
Sequencing library was prepared using NEBNext Ultra Directional RNA Library Prep Kit for Illumina (New England Biolabs) according to manufacturer's instructions.


title: tsc_rip_input_rep5


title: RAD21 ChIP, NIPBLwt

value: NIPBLwt


title: PFC_11

description: Library name: PFC_11

tag: Sex


title: NIPBLmut, rep1


title: PFC_10

description: Library name: PFC_10

value: female

tag: region


title: tsc_rip_input_rep2

tag: genotype


title: tsc_rip_input_rep3


title: tsc_rip_rabbit_igg_rep2

description:
RIP
igg_ns.wig.gz
igg_ps.wig.gz
igg_fc_rpm_2igg_2reps.txt

value: 10ug Invitrogen #026102


title: PFC_9

description: Library name: PFC_9

value: brain


title: PFC_8

description: Library name: PFC_8

value: neuron


title: DMS-MaPseq, dst1∆, dms treated, biol rep 3

first: Leonard

last: Schärfen

address: 368 Plantation Street

department: RNA Therapeutics Institute

institute: UMass Chan Medical School

data_processing:
*library strategy: CoSTseq -or- DMS-MaPseq
The modular CoSTseq data analysis pipeline, implemented including all custom sub modules in Snakemake, is available at https://github.com/NeugebauerLab/CoSTseq. Raw paired-end reads were quality filtered and trimmed with fastp and then aligned to the reference genome with STAR, without generating new splice junctions. Ribosomal RNA was separated based on alignment coordinates and both mRNA and rRNA were deduplicated separately with UMICollapse.
Assembly: sacCer3
Supplementary files format and content: _counts.pkl files contain count data that can be loaded using CoSTseq data processing tools or the "pickle" Python package. Each file includes four lists specifying number of mutations and coverage for each genomic position for both strands: mutation count forward strand, mutation count reverse strand, coverage forward strand, coverage reverse strand. Each list contains 18 numpy arrays: 16 for each chromosome, one for mitochondrial DNA, one for the KanMX cassette used to obtain KO strains.
Supplementary files format and content: _counts_rrna.pkl files contain count data for rrna only that can be loaded using CoSTseq data processing tools or the "pickle" Python package. Each file includes two numpy arrays specifying number of mutations and coverage for each rRNA position.

organism: Saccharomyces cerevisiae

source_name: BY4741 dst1::kanMX

growth_protocol: Liquid cultures from individual colonies of Saccharomyces cerevisiae BY4741 were grown in liquid YPAD medium to an OD600 around 0.6 at 30 ºC with 200 rpm shaking. YKW100 was grown in YPA Gal/Raf and then shifted to YPAG or Gal/Raf.

extract_protocol:
Cells were pelleted at 3,500×g and washed with stop buffer without isoamyl alcohol, then resuspended in 600 µl lysis buffer (45 mM NaOAc, 6 mM EDTA, pH 5.5) with 40 µl 20% SDS added after resuspension. The mixture was then incubated at 65 ºC for 30 s with vigorous shaking, added to 650 µl hot (65 ºC) acid-phenol:chloroform:IAA 125:24:1, pH 4.5 and incubated for 2 min at 65 ºC with shaking. Samples were placed on ice for 5 min, then spun at 20,000×g at room temperature. The aqueous layer was subsequently again extracted with acid-phenol, then with chloroform and RNA was precipitated with isopropanol.
CoSTseq: Approximately 80 µg of input RNA were incubated with Dynabeads MyOne Streptavidin C1 (Invitrogen) according to the manufacturer’s recommendations. The supernatant of the pulldown was retained for mRNA isolation. Beads were washed twice with high salt buffer (2 M NaCl, 50 mM Tris⋅HCl pH 7.4, 0.5% Triton X-100), once with binding buffer (300 mM NaCl, 10 mM Tris⋅HCl pH 7.4, 0.1% Triton X-100), and once with low salt buffer (5 mM Tris⋅HCl pH 7.4, 0.1% Triton X-100); 500 µl each. Nascent RNA was extracted from beads twice using Trizol and chloroform and resuspended in 3.75 µl H2O. Reverse transcription was performed in a 10 µl volume with an optimized KCl based buffer made of 75 mM KCl, 3 mM MgCl2, 50 mM Tris⋅HCl pH 8.3 and 0.5 µl RNase inhibitor using the GsI2c-MBP fusion protein (commercially TGIRT-III), which was purified as described previously. RT reactions were pre-incubated with 100 nM annealed DNA/RNA heteroduplex adapter with a single G overhang to capture the biotin-C at the 3’-end (GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTG, rArArGrArUrCrGrGrArArGrArGrCrArCrArCrGrUrCrUrGrArArCrUrCrCrArGrUrCrArC/3SpC3/) for 30 min at room temperature. Then 1.25 µM dNTP were added and the reaction was incubated at 25 °C for 10 min, 42 °C for 10 min, 50 °C for 10 min, 55 °C for 10 min, 60 °C for 30 min, 65 °C for 20 min, and 75 °C for 15 min. 1 µl of RNase H (NEB) was added and the sample incubated at 37 ºC for 30 min, following clean-up using MinElute PCR Purification kit (Qiagen). The 5’-end adapter (/5Phos/NNNNNNNAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT/3SpC3/) was first adenylated using Mth RNA Ligase (NEB), purified using Oligo Clean & Concentrator (Zymo Research) and then ligated to cDNA using Thermostable 5’ App DNA/RNA Ligase (NEB) according to the manufacturer’s instructions. Adapter ligated cDNA was again cleaned using spin column purification. PCR was carried out with KAPA HiFi HotStart polymerase (Roche) using Illumina TruSeq index primers (i5: AATGATACGGCGACCACCGAGATCTACACXXXXXXXXACACTCTTTCCCTACACGACG, i7: CAAGCAGAAGACGGCATACGAGATXXXXXXXXGTGACTGGAGTTCAGACGTGTG) for 16-20 cycles. Final libraries were size selected in the 200-1,000 bp range by cutting from a polyacrylamide gel. DMS-MaPseq: RNA was precipitated from the supernatant of the biotin pulldown using ethanol. 50 µg were used as input for the Dynabeads Oligo(dT)25 mRNA purification kit (Invitrogen) and mRNA was purified according to the manufacturer’s instructions. RNA was then fragmented using 10 mM ZnCl2 at 94 ºC for 55 s and precipitated using isopropanol. End repair using T4 PNK was performed according to the manufacturer’s instructions. Fragmented mRNA was then size selected by differential silica binding with RNA Clean & Concentrator-5 spin columns (Zymo Research) using 33% ethanol. RT was carried out as described above, except the DNA strand contained a random overhang (N) instead of a G. Adapter ligation, PCR and sequencing were performed as described above. Mature rRNA DMS-MaPseq was carried out by fragmentation and library preparation without any prior enrichment steps.

treatment_protocol: 2.8 ml culture were removed, spun down, and washed with cold PBS. The pellet was resuspended in 10 ml cold 0.5% sarkosyl and incubated on ice for 20 min. Cells were then pelleted at 400×g. Run-on reactions were performed in a 300 µl volume with 25 µM biotin-11-CTP (Jena Biosciences), 125 µM each ATP, UTP and GTP, 0.5% sarkosyl, 20 mM Tris⋅HCl pH 7.4, 200 mM KCl, 5 mM MgCl2 and 2 mM dithiothreitol at 30 ºC for 2 min with mild shaking. 200 µl prewarmed 2.5x structure probing buffer (750 mM Bicine⋅KOH pH 8.0, 500 mM CH3CO2K pH 8.0, 12.5 mM MgCl2) and 25 µl DMS were added and incubated for 4 min at 30 ºC with mild shaking. The reaction was stopped by the addition of 1 ml ice cold stop buffer (20 mM Tris⋅HCl pH 7.4, 200 mM KCl, 4.29 M 2-mercaptoethanol, 50% isoamyl alcohol).

value: Saccharomyces cerevisiae

value: a his3{delta}1 leu2{delta}0 met15{delta}0 ura3{delta}0 dst1::kanMX

value: DMS


title: PFC_6

description: Library name: PFC_6

data_processing:
For Rattus norvegicus samples: Quality control was performed by FastQC, cutadapt was used to trim low quality and over-represented sequences. STAR was used for alignment with ensembl reference (release 104) and RSEM is used for expression calculation.
For Mus musculus samples: Quality control was performed by Fastqc. Perform trimming by trimmomatic, allign the sequence using Hisat2 with Ensembl reference and read count with Hiseq
Assembly: Mus musculus:GRCm38 ; Rattus norvegicus: Rnor_6.0(Ensembl release 104)
Supplementary files format and content: mouse_raw_counts.csv: processed count data for Mus musculus samples.
Supplementary files format and content: rat_raw_counts_all.csv: processed count data for Rattus norvegicus samples.

value: Sal

value: PFC


title: DMS-MaPseq, dst1∆, dms treated, biol rep 2


title: tsc_rip_rabbit_igg_rep1

value: Trophoblast stem cells

value: Wildtype


title: tsc_rip_goat_igg_rep2

value: 10ug Invitrogen #026202


title: NIPBLwt, rep1


title: NIPBLwt, rep2


title: PFC_7

description: Library name: PFC_7

value: male


title: PFC_14

description: Library name: PFC_14

value: Mor + 2W


title: DMS-MaPseq, dst1∆, dms treated, biol rep 1


title: tsc_rip_input_rep1


title: PFC_1

description: Library name: PFC_1


title: tsc_rip_mouse_igg_rep2

value: 10ug Invitrogen #026502


title: PFC_5

description: Library name: PFC_5


title: PFC_2

description: Library name: PFC_2


title: CoSTseq, dst1∆, dms treated, biol rep 2


title: tsc_rip_suz12_rep2

description:
RIP
suz12_ns.wig.gz
suz12_ps.wig.gz
suz12_fc_rpm_2igg_2reps.txt

value: 10ug Cell Signaling #3737


title: tsc_rip_mouse_igg_rep1


title: PFC_3

description: Library name: PFC_3


title: CoSTseq, dst1∆, dms treated, biol rep 1


title: tsc_rip_suz12_rep1


title: tsc_rip_goat_igg_rep1


title: DMS-MaPseq, rRNA, dbp7∆, dms treated, biol rep 3

data_processing:
The modular CoSTseq data analysis pipeline, implemented including all custom sub modules in Snakemake, is available at https://github.com/NeugebauerLab/CoSTseq. Raw paired-end reads were quality filtered and trimmed with fastp and then aligned to the reference genome with STAR, without generating new splice junctions. Ribosomal RNA was separated based on alignment coordinates and both mRNA and rRNA were deduplicated separately with UMICollapse.
Assembly: sacCer3
Supplementary files format and content: _counts.pkl files contain count data that can be loaded using CoSTseq data processing tools or the "pickle" Python package. Each file includes four lists specifying number of mutations and coverage for each genomic position for both strands: mutation count forward strand, mutation count reverse strand, coverage forward strand, coverage reverse strand. Each list contains 18 numpy arrays: 16 for each chromosome, one for mitochondrial DNA, one for the KanMX cassette used to obtain KO strains.
Supplementary files format and content: _counts_rrna.pkl files contain count data for rrna only that can be loaded using CoSTseq data processing tools or the "pickle" Python package. Each file includes two numpy arrays specifying number of mutations and coverage for each rRNA position.
Library strategy: DMS-MaPseq

source_name: BY4741 dbp7::kanMX

growth_protocol: Liquid cultures from individual colonies of Saccharomycescerevisiae BY4741 were grown in liquid YPAD medium to an OD600 around 0.6 at 30ºC with 200rpm shaking. YKW100 was grown in YPA Gal/Raf and then shifted to YPAG or Gal/Raf.

extract_protocol:
Cells were pelleted at 3,500×g and washed with stop buffer without isoamyl alcohol, then resuspended in 600µl lysis buffer (45mM NaOAc, 6mM EDTA, pH 5.5) with 40µl 20% SDS added after resuspension. The mixture was then incubated at 65ºC for 30s with vigorous shaking, added to 650µl hot (65ºC) acid-phenol:chloroform:IAA 125:24:1, pH 4.5 and incubated for 2min at 65ºC with shaking. Samples were placed on ice for 5min, then spun at 20,000×g at room temperature. The aqueous layer was subsequently again extracted with acid-phenol, then with chloroform and RNA was precipitated with isopropanol.
CoSTseq: Approximately 80µg of input RNA were incubated with Dynabeads MyOne Streptavidin C1 (Invitrogen) according to the manufacturer’s recommendations. The supernatant of the pulldown was retained for mRNA isolation. Beads were washed twice with high salt buffer (2M NaCl, 50mM Tris⋅HCl pH 7.4, 0.5% Triton X-100), once with binding buffer (300mM NaCl, 10mM Tris⋅HCl pH 7.4, 0.1% Triton X-100), and once with low salt buffer (5mM Tris⋅HCl pH 7.4, 0.1% Triton X-100); 500µl each. Nascent RNA was extracted from beads twice using Trizol and chloroform and resuspended in 3.75µl H2O. Reverse transcription was performed in a 10µl volume with an optimized KCl based buffer made of 75mM KCl, 3mM MgCl2, 50mM Tris⋅HCl pH 8.3 and 0.5µl RNase inhibitor using the GsI2c-MBP fusion protein (commercially TGIRT-III), which was purified as described previously. RT reactions were pre-incubated with 100nM annealed DNA/RNA heteroduplex adapter with a single G overhang to capture the biotin-C at the 3’-end (GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTG, rArArGrArUrCrGrGrArArGrArGrCrArCrArCrGrUrCrUrGrArArCrUrCrCrArGrUrCrArC/3SpC3/) for 30min at room temperature. Then 1.25µM dNTP were added and the reaction was incubated at 25°C for 10min, 42°C for 10min, 50°C for 10min, 55°C for 10min, 60°C for 30min, 65°C for 20min, and 75°C for 15min. 1µl of RNase H (NEB) was added and the sample incubated at 37ºC for 30min, following clean-up using MinElute PCR Purification kit (Qiagen). The 5’-end adapter (/5Phos/NNNNNNNAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT/3SpC3/) was first adenylated using MthRNALigase (NEB), purified using Oligo Clean & Concentrator (Zymo Research) and then ligated to cDNA using Thermostable5’AppDNA/RNALigase (NEB) according to the manufacturer’s instructions. Adapter ligated cDNA was again cleaned using spin column purification. PCR was carried out with KAPAHiFiHotStart polymerase (Roche) using Illumina TruSeq index primers (i5: AATGATACGGCGACCACCGAGATCTACACXXXXXXXXACACTCTTTCCCTACACGACG, i7: CAAGCAGAAGACGGCATACGAGATXXXXXXXXGTGACTGGAGTTCAGACGTGTG) for 16-20 cycles. Final libraries were size selected in the 200-1,000 bp range by cutting from a polyacrylamide gel. DMS-MaPseq: RNA was precipitated from the supernatant of the biotin pulldown using ethanol. 50 µg were used as input for the Dynabeads Oligo(dT)25 mRNA purification kit (Invitrogen) and mRNA was purified according to the manufacturer’s instructions. RNA was then fragmented using 10 mM ZnCl2 at 94 ºC for 55 s and precipitated using isopropanol. End repair using T4 PNK was performed according to the manufacturer’s instructions. Fragmented mRNA was then size selected by differential silica binding with RNA Clean & Concentrator-5 spin columns (Zymo Research) using 33% ethanol. RT was carried out as described above, except the DNA strand contained a random overhang (N) instead of a G. Adapter ligation, PCR and sequencing were performed as described above. Mature rRNA DMS-MaPseq was carried out by fragmentation and library preparation without any prior enrichment steps.

treatment_protocol: 2.8ml culture were removed, spun down, and washed with cold PBS. The pellet was resuspended in 10ml cold 0.5% sarkosyl and incubated on ice for 20min. Cells were then pelleted at 400×g. Run-on reactions were performed in a 300µl volume with 25µM biotin-11-CTP (Jena Biosciences), 125µM each ATP, UTP and GTP, 0.5% sarkosyl, 20mM Tris⋅HCl pH 7.4, 200mM KCl, 5mM MgCl2 and 2mM dithiothreitol at 30ºC for 2min with mild shaking. 200µl prewarmed 2.5x structure probing buffer (750mM Bicine⋅KOH pH 8.0, 500mM CH3CO2K pH 8.0, 12.5mM MgCl2) and 25µl DMS were added and incubated for 4min at 30ºC with mild shaking. The reaction was stopped by the addition of 1ml ice cold stop buffer (20mM Tris⋅HCl pH 7.4, 200mM KCl, 4.29M 2-mercaptoethanol, 50% isoamyl alcohol).

value: BY4741 dbp7::kanMX

value: a his3{delta}1 leu2{delta}0 met15{delta}0 ura3{delta}0 dbp7::kanMX


title: tsc_rip_rybp_rep2

description:
RIP
rybp_ns.wig.gz
rybp_ps.wig.gz
rybp_fc_rpm_2igg_2reps.txt

value: 10ug Millipore Sigma AB3637


title: DMS-MaPseq, rRNA, dbp7∆, dms treated, biol rep 2


title: ATP 3

city: Bonn

first: Günther

last: Weindl

address: Gerhard-Domagk-Str. 3

department: Pharmaceutical Institute

country: Germany

institute: University of Bonn

data_processing:
Sequence reads were trimmed using cutadapt.
Alignment of the trimmed reads against human genime (GRCh38) was performed using STAR.
Reads were assigned to genes using FeatureCounts.
Assembly: GRCh38
Supplementary files format and content: Excel files include raw counts for each sample
Supplementary files format and content: Excel files include comparison of samples and gene names

source_name: PMA-differentiated THP-1 cells

growth_protocol: THP-1 macrophages were derived from THP-1 monocytes using PMA (Phorbol 12-myristate 13-acetate). The cells were cultured in RPMI containing 10% fetal bovine serum (FBS), 2 mM L-glutamine and antibiotics (Penicillin/Streptomycin) in humidified atmosphere with 5% CO2 at 37 °C.

extract_protocol:
RNA was harvested using innuPREP RNA Mini Kit 2.0 (Analytik Jena)
RNA libaries for RNA-seq were prepared using QuantSeq 3'-mRNA Libraray Prep Kit (Lexogen).

treatment_protocol: THP-1 macrophages were incubated with medium (RPMI) with or without 5 mM of Ap4 or ATP for 3 h.

value: PMA-differentiated THP-1 cells

value: macrophages

value: ATP 5 mM


title: DpC_5

city: Durham

first: KUO

last: DU

address: 905 SOUTH LASALLE STREET, Room 1063

department: Medicine

institute: Duke Universty

description: Library name: Sample10

data_processing:
RNA-seq data were processed using the fastp toolkit to trim low-quality bases and Illumina sequencing adapters from the 3’ end of the reads. Only reads that were ≥20nt after trimming were kept for further analysis. Reads were mapped to mice genome reference combining the GRCm38. STAR v2.7.5 was used to align reads to reference genome. Gene count was quantified by featureCounts v2.0.1.
Assembly: GRCm38 and mm10
Supplementary files format and content: tsv and txt

source_name: Liver

extract_protocol:
RNA was harvested using Trizol (Invitrogen)
RNA samples were sequenced by Novogene Corporation following standard construction protocol. In brief, RNA degradation and contamination were monitored on 1% agarose gels. RNA purity was checked using a NanoPhotometer Spectrophotometer (IMPLEN). RNA integrity and quantity were assessed using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies). Sequencing libraries were generated using the NEB Next Ultra RNA Library Prep Kit for Illumina (NEB) according to the manufacturer’s recommendations and index codes were added to attribute sequences to each sample. Clustering of index-coded samples was performed on an Illumina NovaSeq sequencer

value: Liver

value: DpC


title: DpC_3

description: Library name: Sample8


title: DMS-MaPseq, rRNA, wildtype, dms treated, biol rep 3

value: a his3{delta}1 leu2{delta}0 met15{delta}0 ura3{delta}0

value: DMS


title: tsc_rip_ring1b_rep2

description:
RIP
ring1b_ns.wig.gz
ring1b_ps.wig.gz
ring1b_fc_rpm_2igg_2reps.txt

value: 10ug Cell Signaling #5694

value: Trophoblast stem cells


title: CoSTseq, dst1∆, dms treated, biol rep 3

value: Saccharomyces cerevisiae

value: a his3{delta}1 leu2{delta}0 met15{delta}0 ura3{delta}0 dst1::kanMX


title: Ap4 3

value: Ap4 5 mM


title: DMS-MaPseq, rRNA, dbp7∆, dms treated, biol rep 1


title: tsc_rip_rybp_rep1

value: Wildtype


title: Control 3

value: Medium (RPMI)


title: DpC_2

description: Library name: Sample7


title: ATP 2


title: DMS-MaPseq, rRNA, wildtype, dms treated, biol rep 2


title: DpC_1

description: Library name: Sample6


title: tsc_rip_ring1b_rep1


title: Ap4 2


title: Control 2


title: Veh_5

description: Library name: Sample5

value: Vehicle


title: Veh_4

description: Library name: Sample4


title: DpC_4

description: Library name: Sample9


title: tsc_rip_mtf2_rep1

description:
RIP
mtf2_ns.wig.gz
mtf2_ps.wig.gz
mtf2_fc_rpm_2igg_2reps.txt

value: 10ug Proteintech 16208-1-AP


title: Veh_3

description: Library name: Sample3


title: CoSTseq, YKW100, untreated, glucose, biol rep 2

value: a ura3-52 his3-{delta}leu2 lys2-801amber trp1-{delta}63 u3a{delta} UASGAL:U3A::URA3 u3b{delta}::LEU2

value: untreated


title: Veh_2

description: Library name: Sample2


title: CoSTseq, YKW100, untreated, galactose, biol rep 2

data_processing:
The modular CoSTseq data analysis pipeline, implemented including all custom sub modules in Snakemake, is available at https://github.com/NeugebauerLab/CoSTseq. Raw paired-end reads were quality filtered and trimmed with fastp and then aligned to the reference genome with STAR, without generating new splice junctions. Ribosomal RNA was separated based on alignment coordinates and both mRNA and rRNA were deduplicated separately with UMICollapse.
Assembly: sacCer3
Supplementary files format and content: _counts.pkl files contain count data that can be loaded using CoSTseq data processing tools or the "pickle" Python package. Each file includes four lists specifying number of mutations and coverage for each genomic position for both strands: mutation count forward strand, mutation count reverse strand, coverage forward strand, coverage reverse strand. Each list contains 18 numpy arrays: 16 for each chromosome, one for mitochondrial DNA, one for the KanMX cassette used to obtain KO strains.
Supplementary files format and content: _counts_rrna.pkl files contain count data for rrna only that can be loaded using CoSTseq data processing tools or the "pickle" Python package. Each file includes two numpy arrays specifying number of mutations and coverage for each rRNA position.
Library strategy: CoSTseq


title: CoSTseq, YKW100, untreated, glucose, biol rep 1


title: CoSTseq, YKW100, untreated, galactose, biol rep 1


title: Ap4 1

value: PMA-differentiated THP-1 cells

value: macrophages


title: tsc_rip_jarid2_rep2

description:
RIP
jarid2_ns.wig.gz
jarid2_ps.wig.gz
jarid2_fc_rpm_2igg_2reps.txt


title: Control 1


title: tsc_rip_hnrnpk_rep2

description:
RIP
hnk_ns.wig.gz
hnk_ps.wig.gz
hnrnpk_fc_rpm_2igg_2reps.txt

value: 10ug Santa Cruz sc-28380


title: CoSTseq, YKW100, dms treated, glucose, biol rep 3


title: tsc_rip_hnrnpk_rep1


title: tsc_rip_mtf2_rep2


title: 98_12d_HDF3_Con_F

city: Rochester

first: Fathima

last: Athar

state: New York

address: 500 Joseph C Wilson Blvd.

department: Biology

institute: University of Rochester

data_processing:
Fastp (0.23.4): Default parameters Salmon (v1.5.1): salmon quant --gcBias -l A
Assembly: GCA_003508835.1 (Eonycteris spelaea), EptFus1.0_HiC (DNA Zoo Consortium, Eptesicus fuscus), GCA_004027435.1 (Artibeus jamaicensis), Myoluc2.0_HiC (DNA Zoo Consortium, Myotis lucifugus), GCF_000001635.27 (Mus musculus), and GCA_000001405.29 (Homo sapiens)
Supplementary files format and content: Row count and matrix

source_name: Fibroblast

extract_protocol:
Total RNA was extracted from irradiated or non-irradiated fibroblasts from four bat species, laboratory mice, wild-caught mice and humans using PureLinkTM RNA Mini Kit (Thermo Fisher Scientific) following manufacturer’s instructions.
NGS-TruSeq Stranded mRNA libraries were generated and sequenced with Illumina NovaSeq 6000 single-end 75 bp sequencing at the University of Rochester Genomics Research Center.

value: Fibroblast

value: wildtype


title: 3 μm_confine_rep3

city: Suzhou

first: Peng

last: Shi

address: Renai street,199

institute: Soochow University

description: Library name: Sample6

data_processing:
Adaptor and low-quality reads were removed using trim-galore (0.6.10).
The data was aligned to the murine genome using hisat2 (2.1.0).
Alignment files in SAM/BAM format were processed using samtools (1.5), including inputting and outputting alignment files, sorting, merging and index.
FeatureCounts was used to count different genomic features such as genes, exons, promoters, genosomes, genome boxes, and chromosome locations.
Assembly: mm10
Supplementary files format and content: table-delimited txt file includes raw counts for each samples

source_name: Mouse Embryonic Fibroblasts

growth_protocol: Cells were cultured in Dulbecco’s modified Eagle medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin and 100 mg/mL streptomycin at 37 °C with 5% CO2.

extract_protocol:
Total RNA was extracted using Trizol Reagent (Invitrogen, 15596018). Total RNA was qualified and quantified as follows: (1) RNA purity and concentration were examined using NanoDrop 2000; (2) RNA integrity and quantity were measured using the Agilent 2100/4200 system.
The mRNA was purified from total RNA using polyT and then fragmented into 300-350 bp fragments. The first strand cDNA was reverse-transcribed using fragmented RNA and dNTPs (dATP, dTTP, dCTP and dGTP) and second strand cDNA was synthesized using DNA polymerase I and dNTPs (dATP, dUTP, dCTP and dGTP). Remaining overhangs of double-strand cDNA were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3’ ends of DNA fragments, sequencing adaptors were ligated to the cDNA and the library fragments were purified. The template without U was enriched by PCR, and the PCR product was purified to obtain the final library.

treatment_protocol: MEF cells were seeded on the upper surface f transwell with 3 μm pore size. After 15 hours, transmigrated cells were collected.

value: Mouse Embryonic Fibroblasts

value: MEFs

value: WT

value: Confinement


title: CoSTseq, YKW100, dms treated, glucose, biol rep 2

value: DMS


title: 3 μm_confine_rep2

description: Library name: Sample5


title: tsc_rip_ezh2_rep2

description:
RIP
ezh2_ns.wig.gz
ezh2_ps.wig.gz
ezh2_fc_rpm_2igg_2reps.txt

value: 10ug Gift from R. Bonasio

value: Trophoblast stem cells


title: DMS-MaPseq, rRNA, wildtype, dms treated, biol rep 1

value: a his3{delta}1 leu2{delta}0 met15{delta}0 ura3{delta}0


title: 97_HDF3_Con_P


title: tsc_rip_ezh2_rep1

value: Wildtype


title: CoSTseq, YKW100, dms treated, glucose, biol rep 1


title: 96_12d_HDF2_20Gy


title: ATP 1

value: ATP 5 mM


title: tsc_rip_jarid2_rep1

value: 10ug Cell Signaling #135945


title: CoSTseq, YKW100, dms treated, galactose, biol rep 3

value: a ura3-52 his3-{delta}leu2 lys2-801amber trp1-{delta}63 u3a{delta} UASGAL:U3A::URA3 u3b{delta}::LEU2


title: 3 μm_confine_rep1

description: Library name: Sample4


title: Veh_1

description: Library name: Sample1

value: Liver

value: Vehicle


title: Control-rep3

description: Library name: Sample3

value: Control


title: tsc_rip_epop_rep2

description:
RIP
epop_ns.wig.gz
epop_ps.wig.gz
epop_fc_rpm_2igg_2reps.txt

value: 10ug Active Motif 61753


title: tsc_rip_epop_rep1


title: 95_12d_HDF2_10Gy

value: Fibroblast

value: wildtype


title: CoSTseq, YKW100, dms treated, galactose, biol rep 2


title: Control-rep2

description: Library name: Sample2

value: Mouse Embryonic Fibroblasts

value: MEFs


title: 94_12d_HDF2_Con_F


title: 3-day 10ng/mL dox-treated Xist-RMCE clone 4 RNA

description:
A pooled WIG file was generated using reads from the two or more biological replicates/clones during the STAR alignment
xist-rmce_3d_10dox_pooled_rna_mm10_neg.wig
xist-rmce_3d_10dox_pooled_rna_mm10_pos.wig

data_processing:
Sequencing reads (fastq files) were mapped both to the GENCODE vM25 mouse genome (“basic CHR” from https://www.gencodegenes.org/mouse/release_M25.html) [for B6] and to a version of the same genome modified to incorporate CAST single-nucleotide polymorphisms (SNPs) [for CAST]. Mapping was performed with STAR (v2.7.9a), using two-pass mapping and the option “--outFilterMultimapNmax 1” to consider only uniquely mapping reads.
Using a custom Perl script (intersect_reads_snps16.pl) and a file containing a list of CAST SNPs (sangercast07_30_20), aligned reads in the B6 and CAST SAM files were parsed to identify reads clearly originating from either the B6 or CAST allele (i.e. reads that overlap at least 1 B6 or CAST SNP).
For genome browser viewing, strand-specific mm10-aligned reads were separated using samtools: samtools view -Sq 30 (negative) -F 0x10 or (positive) -f 0x10
Assembly: mm10
Supplementary files format and content: WIG files were created using a custom perl script. Per dox condition/genotype, a pooled WIG file was generated using reads from the two or more biological replicates/clones during the STAR alignment.

extract_protocol:
RMCE ESCs or MEF feeder-free TSCs were washed twice with ice-cold PBS and harvested with 1 mL TRIzol (Thermo Fisher 15596026). Total RNA was prepared via standard TRIzol-chlorofom extraction, per manufacturer’s instructions, except for the addition of 4μL 5μg/μL linear acrylamide (Thermo Fisher AM9520) to promote precipitation in isopropanol. RNA pellets were fully resuspended in RNase-free water, RNA concentrations were measured via Qubit 2.0 fluorometer (RNA high sensitivity kit, Thermo Fisher Q32852), and integrity of RNA was confirmed by visualizing rRNA bands on an agarose gel. Working stocks of 100ng/μL total RNA were prepared, and all RNA samples were stored at -80°C.
Libraries for RNA sequencing were prepared using the KAPA RNA HyperPrep Kit with RiboErase (HMR) (Roche 08098140702) and KAPA Unique Dual-Indexed Adapters (Roche 08861919702) or TruSeq adapters, with each library’s input consisting of 9μL 100ng/μL total RNA (900 ng) mixed with 1μL of a 1:250 dilution of ERCC RNA spike-in controls (Thermo Fisher 4456740). RNA fragmentation was performed at 94°C for 6min. Libraries were pooled and sequenced on an Illumina NextSeq500 platform with a 75-cycle NextSeq 500/550 High Output Kit v2.5 (Illumina 20024906).

value: RMCE Embryonic Stem Cells

value: 3 days with 10ng/mL doxycycline

value: Xist-RMCEs


title: 93_HDF2_Con_P


title: 99_12d_HDF3_10Gy


title: CoSTseq, YKW100, dms treated, galactose, biol rep 1

value: DMS


title: Control-rep1

description: Library name: Sample1

value: WT


title: DMS-MaPseq, stm1∆, dms treated, biol rep 3

source_name: BY4741 stm1::kanMX

value: BY4741 stm1::kanMX

value: a his3{delta}1 leu2{delta}0 met15{delta}0 ura3{delta}0 stm1::kanMX


title: vdj_np396_5

city: Freising

first: Gustavo

middle: P.

last: de Almeida

state: Bavaria

address: Liesel-Beckmann-Str. 1

department: Division of Animal Physiology and Immunology

institute: Technical University of Munich

description: scRNA-Seq + TCR (10x Genomics)

data_processing:
References refdata-cellranger-arc-mm10-2020-A-2.0.0.tar.gz and refdata-cellranger-vdj-GRCm38-alts-ensembl-7.0.0.tar.gz were retrieved from 10xgenomics.com and used to align the reads of the gene expression and TCR assay with cellranger multi (v7.1.0).
Assembly: GRCm38.p6 (release 98) - GENCODE Release M23
Supplementary files format and content: Annotated data matrix (anndata) object saved in h5ad-file format. The file contains the preprocessed, normalized and annotated count data of all samples.

source_name: spleen

extract_protocol:
Spleens were harvested from infected mice and macerated through a 100µm Nytex filter, washed with DMEM and erythrocyte lysis was performed using a hypotonic ACK medium. Lysis was stopped by adding DMEM containing 10% FCS. Cells were counted using a Neubauer counting chamber  stained with flurorescently labelled antibodies against CD8a,  CD44 as well as a fixable live/dead dye and multimers loaded with either gp33 or np396 and conjugated to different flurochromes. Single, live reporter+ CD8 T cells reacting to either of the multimers were sorted and used for library generation.
Gene expression and T-cell receptor V(D)J libraries were prepared by using the Chromium Next GEM Single Cell 5’ Reagent Kit v2 (10X Genomics), Chromium Single Cell Mouse TCR Amplification Kit (10X Genomics) and Chromium Next GEM Chip K Single Cell Kit (10X Genomics) following the manufacturer’s protocol (CG000331 Rev E). Dual Index Kit TT Set A (10X Genomics) was used for multiplexing (i7 and i5 index read, 10bp). The samples were sequenced in a paired-end run (read 1: 26bp, read 2: 90bp) on a NovaSeq6000 platform using S1 v1.5 (100 cycles) sequencing kits (Illumina). Bcl2fastq software (v2.20.0.422) was used for demultiplexing and generating .fastq files.  The libraries were sequenced in a paired-end run (read 1: 26bp, read 2: 90bp) on a NovaSeq6000 platform using S1 v1.5 (100 cycles) sequencing kits (Illumina). Bcl2fastq software (v2.20.0.422) was used for demultiplexing and generating .fastq files.

treatment_protocol: TCF1 reporter mice were infected with LCMV Armstrong, splenocytes were harvested 4 weeks post infection.

value: spleen

value: CD8+ T cells

tag: epitope

tag: phenotype

tag: time point

value: day 30 post infection

value: LCMV Armstrong

tag: library type

value: VDJ


title: 3-day 10ng/mL dox-treated Xist-RMCE clone 1 RNA

tag: cell line


title: DMS-MaPseq, stm1∆, dms treated, biol rep 2


title: 3-day 1000ng/mL dox-treated Xist-RMCE ESC clone 4 H3K27me3

value: 3 days with 1000ng/mL doxycycline

tag: chip antibody

value: Tri-Methyl-Histone H3 (Lys27) (C36B11) (H3K27me3) (Cell Signaling, cat #:9733)


title: vdj_np396_3

description: scRNA-Seq + TCR (10x Genomics)


title: vdj_np396_2

description: scRNA-Seq + TCR (10x Genomics)


title: 3-day 1000ng/mL dox-treated Xist-RMCE ESC clone 1 H3K27me3


title: 90_24h_HDF86F_20Gy

value: Fibroblast

value: wildtype


title: DMS-MaPseq, stm1∆, dms treated, biol rep 1


title: DMS-MaPseq, dbp7∆, dms treated, biol rep 3

value: BY4741 dbp7::kanMX

value: a his3{delta}1 leu2{delta}0 met15{delta}0 ura3{delta}0 dbp7::kanMX


title: 3-day 10ng/mL dox-treated Xist-RMCE ESC clone 5 H3K27me3

value: RMCE Embryonic Stem Cells

value: 3 days with 10ng/mL doxycycline


title: 92_12d_HDF1_20Gy


title: vdj_gp33_3

description: scRNA-Seq + TCR (10x Genomics)


title: 89_HDF1_Con_P


title: vdj_gp33_5

description: scRNA-Seq + TCR (10x Genomics)


title: 3-day 10ng/mL dox-treated Xist-RMCE ESC clone 4 H3K27me3

value: Xist-RMCEs


title: 89_24h_HDF86F_10Gy


title: DMS-MaPseq, dbp7∆, dms treated, biol rep 1

value: DMS


title: 3-day 10ng/mL dox-treated Xist-RMCE ESC clone 3 H3K27me3


title: DMS-MaPseq, dbp7∆, dms treated, biol rep 2


title: DMS-MaPseq, dbp3∆, dms treated, biol rep 3

source_name: BY4741 dbp3::kanMX

value: BY4741 dbp3::kanMX

value: a his3{delta}1 leu2{delta}0 met15{delta}0 ura3{delta}0 dbp3::kanMX


title: 90_12d_HDF1_Con_F


title: 3-day 10ng/mL dox-treated Xist-RMCE ESC clone 1 H3K27me3


title: gex_np396_5

description: scRNA-Seq + TCR (10x Genomics)

value: spleen

value: GEX


title: vdj_gp33_2

description: scRNA-Seq + TCR (10x Genomics)

value: CD8+ T cells

value: day 30 post infection


title: DMS-MaPseq, dbp3∆, dms treated, biol rep 2


title: 88_24h_HDF86F_Con


title: gex_np396_3

description: scRNA-Seq + TCR (10x Genomics)

value: LCMV Armstrong


title: Untreated Xist-RMCE ESC clone 5 H3K27me3

value: Untreated


title: 87_24h_HDF70F_20Gy


title: 91_12d_HDF1_10Gy


title: 86_24h_HDF70F_10Gy


title: gex_np396_2

description: scRNA-Seq + TCR (10x Genomics)


title: DMS-MaPseq, dbp3∆, dms treated, biol rep 1