title: Control 1 mock


title: Covid-19-Exo_4-26_89

status: Public on May 13 2021

type: SRA

city: Jerusalem

first: Iddo

middle: Z.

last: Ben-Dov

address: Ein Kerem

department: Nephrology and Hypertension

country: Israel

institute: Hadassah Medical Center

library_source: transcriptomic

data_processing:
Standard Illumina base-calling (HiSeq 2500)
Adapter trimming, allignment and annotation on rnaworld (PMID: 22836126)
Genome_build: hg19
Supplementary_files_format_and_content: Excel files showing miRNA read counts and sample metadata

molecule: total RNA

organism: Homo sapiens

source_name: exosomes immunopurified from serum (exo-CATCHER protocol)

extract_protocol:
Total RNA extraction from serum, plasma or pulled exosomes using miRNeasy Serum/Plasma kit (Qiagen, # 217184)
In-house small RNA library preparation (as described in PMID 28335433)

tag: disease severity

value: Severe

tag: library preparation

value: In-house

tag: 3' adapter sequence

value: TAACGTCGTATGCCGTCTTCTGCTTG


title: Control 1 CMC

city: Glasgow

first: Alison

last: Galloway

address: Garscube Estate, Switchback Road

country: United Kingdom

institute: Beatson CRUK

data_processing:
Libraries from the first experiment comparing antibodies were sequenced on a MiSeq with a v3 150 cycle kit in paired-end mode. Libaries from the Rnmt cKO vs control CD4 T cell libraries were sequenced on a  NextSeq 500 with a mid output v2.5 kit (150 cycles) in single end mode.
First the 10nt randomer barcode was extracted using umi_tools, then adapter sequences were trimmed from the reads using cutadapt (AGATCGGAAGAGCACACGTCTGAACTCCAGTCA). For paired end libaries only read one was used since reads were too short for the second read to be advantageous.
Reads were aligned to mm10 with gencode vM12 basic annotation using STAR 2.5.2b, then demultiplexed using umi_tools to remove PCR duplicates.
Secondary alignments and alignments with a MAPQ <20 were removed using samtools.
Bedgraph files were generated using deeptools, options -bs 1 --Offset 1 1  -of bedgraph
Genome_build: Supplementary_Data_1.fa from GSE128947
Supplementary_files_format_and_content: Bed files have the counts of reads starting within the 1bp windows

organism: Mus musculus

source_name: 20 hour activated CD4 T cells

growth_protocol: CD4 T cells were sorted from lymph nodes using an EasySep mouse naive CD4 T cell isolation kit.

extract_protocol:
Total RNA was extracted using trizol.
Libraries were made following the pseudoruridine sequencing protocol published by Schwartz, S. et al 2014. 300ng of total RNA from each replicate was made up in BEU buffer (50mM bicine pH 8.3, 4mM EDTA, 7M urea) for the mock treated sample or BEU buffer plus 0.17M CMC (N-Cyclohexyl-N′-(2-morpholinoethyl)carbodiimide methyl-p-toluenesulfonate) (Sigma). Samples were heated to 37°C for 20 mins, then RNA precipitated by adding 100ul buffer A (0.3M NaOAc, 0.1mM EDTA), 1 ul glycogen and 700ul ethanol and washed 1X in 75% EtOH. Samples were resuspended in buffer A, then precipitated as before with ethanol to remove any remaining CMC. RNA was made up in 50mM sodium bicarbonate pH10.4 and heated to 37°C for 3 hours (this removes unwanted CMC adducts). RNA was precipitated with 100ul buffer A, 1ul glycogen, 700ul Ethanol, and washed in 75% ethanol. RNA was made up in 50mM sodium bicarbonate pH9.2 and heated to 95C for 8 mins to fragment, then onto ice. RNA was precipitated with 200ul buffer A, 1 ul glycogen, 500ul ethanol, and washed in 75% ethanol. RNA was dephosphorylated with FastAP (thermo), and linker ligated with high concentration T4 ligase (NEB). Products were reverse transcribed, then the second linker ligated using the high concentration T4 ligase (NEB). Clean ups between these steps were conducted using myOne silane beads. The libraries were PCR amplified and gel purified, then sequenced with a Nextseq High Output v2.5 kit (75 cycles, single end).

treatment_protocol: CD4 T cells were cultured for 20 hours in T cell culture medium (RPMI + 10% heat inactivated FCS + pen/strep + 50µM 2ME) on anti-CD3/CD28 coated plates prepared by incubating the plates with PBS with 5ug/ml anti-CD3 and 1ug/ml anti-CD28 at 37C for three hours.

tag: genotype

value: Rnmt fl/fl CD4cre -

tag: Sex

value: Female

tag: treatment

value: CMC

tag: cell type

value: CD4 T cells


title: Covid-19-Exo_4-26_84

value: TCTGATCGTATGCCGTCTTCTGCTTG


title: Covid-19-Exo_4-26_83

value: TCTCCTCGTATGCCGTCTTCTGCTTG


title: RPF_Rnmt_cKO_CD4_3

description:
RPF (ribosome protected fragment) RNA
Rnmt cKO
RPFKO3

data_processing:
Library strategy: Ribo-Seq
Libraries were sequenced on an Illumina NextSeq 500 using a high output 2 X 75bp kit.
Quality control in FastQC indicated that read one had failed, therefore only read two was used for alignment.
Prior to alignment, adapter sequences (GATCGTCGGACTGTAGAACT) were removed using cutadapt.
Reads aligning to ribosomal RNA and tRNAs were filtered out using bowtie, then the remaining reads were aligned to mm10 with gencode vM12 basic annotation using STAR 2.5.2b.
EdgeR (Robinson et al., 2010) was used for comparisons between control and Rnmt cKO total RNA and RPF RNAs, and Ribodiff (Zhong et al., 2016) was used to calculate and compare translation efficiencies.
Genome_build: mm10 (GRCm38)
Supplementary_files_format_and_content: *_ReadsPerGene.out.tab: Each file contains a tab-delimited table of reads per gene counted during alignment in STAR. The first column indicates the Ensembl gene id, the second column shows all alignments, the third shows forwards alignments, and the fourth shows reverse alignments (reverse aligned reads are the reads counts to use for this library strategy).
Supplementary_files_format_and_content: Riboseq_analysis.csv: The processed data file contains a comma-delimited table of the fold changes and statistical significance of comparisons between Rnmt cKO and control cells, and reads per million (RPM) of each gene: TE=translation efficiency, RPF=ribosome protected fragment RNA, Total= total cytoplasmic RNA, C= control cells, KO= Rnmt cKO cells.

extract_protocol:
Cells were lysed and the cytoplasmic RNA and ribosome protected fragment RNA prepared using the Illumina TruSeq Ribo Profile (mammalian) kit.
Libraries were prepared using the Illumina TruSeq Ribo Profile (mammalian) kit.

treatment_protocol: CD4 T cells were cultured for 20 hours in T cell culture medium (RPMI + 10% heat inactivated FCS + pen/strep + 50µM 2ME) on anti-CD3/CD28 coated plates prepared by incubating the plates with PBS with 5ug/ml anti-CD3 and 1ug/ml anti-CD28 at 37C for three hours. Prior to lysis, cells were incubated with 0.1mg/ml cycloheximide for 1 minute.

value: Rnmt fl/fl CD4cre +

value: 20 hour activated CD4 T cells


title: RPF_Rnmt_cKO_CD4_2

description:
RPF (ribosome protected fragment) RNA
Rnmt cKO
RPFKO2


title: Covid-19-Exo_4-26_82

value: TCTAGTCGTATGCCGTCTTCTGCTTG


title: Rnmt cKO 2 CMC


title: RPF_Rnmt_cKO_CD4_1

description:
RPF (ribosome protected fragment) RNA
Rnmt cKO
RPFKO1


title: RPF_Control_CD4_3

description:
RPF (ribosome protected fragment) RNA
Control
RPFC3


title: Covid-19-Exo_4-26_81

value: TCGCGTCGTATGCCGTCTTCTGCTTG


title: Covid-19-Exo_4-26_80

value: Mild

value: TCGATTCGTATGCCGTCTTCTGCTTG


title: Covid-19-Exo_4-26_79

value: TCCTATCGTATGCCGTCTTCTGCTTG


title: Covid-19-Exo_4-26_78

value: TCCGTTCGTATGCCGTCTTCTGCTTG


title: Control 2 mock

value: mock


title: Covid-19-Exo_4-26_76

value: In-house

value: TCATCTCGTATGCCGTCTTCTGCTTG


title: Covid-19-Exo_4-26_75

value: TCACTTCGTATGCCGTCTTCTGCTTG


title: RPF_Control_CD4_2

description:
RPF (ribosome protected fragment) RNA
Control
RPFC2

value: Rnmt fl/fl CD4cre -

value: Female


title: Covid-19-Exo_3-26_94

value: Death

value: TGATGTCGTATGCCGTCTTCTGCTTG


title: Total_Rnmt_cKO_CD4_1

description:
Total cytoplasmic RNA
Rnmt cKO
TotalKO1


title: Total_Control_CD4_3

description:
Total cytoplasmic RNA
Control
TotalC3


title: Total_Rnmt_cKO_CD4_3

description:
Total cytoplasmic RNA
Rnmt cKO
TotalKO3


title: RPF_Control_CD4_1

description:
RPF (ribosome protected fragment) RNA
Control
RPFC1


title: Covid-19-Exo_4-26_77

value: TCCACTCGTATGCCGTCTTCTGCTTG


title: Covid-19-Exo_3-26_91

value: Severe

value: TAGAGTCGTATGCCGTCTTCTGCTTG


title: Total_Control_CD4_1

description:
Total cytoplasmic RNA
Control
TotalC1

value: 20 hour activated CD4 T cells


title: Total_Rnmt_cKO_CD4_2

description:
Total cytoplasmic RNA
Rnmt cKO
TotalKO2

value: Rnmt fl/fl CD4cre +


title: Total_Control_CD4_2

description:
Total cytoplasmic RNA
Control
TotalC2


title: Covid-19-Exo_3-26_92

value: TAGGATCGTATGCCGTCTTCTGCTTG


title: Rnmt_cKO_rabbit_IgG_IP

description:
seCLIP library of rabbit IgG control interacting RNA.
Rnmt cKO
ko_L1_pureclip_region.bed

data_processing:
Libraries from the first experiment comparing antibodies were sequenced on a MiSeq with a v3 150 cycle kit in paired-end mode. Libraries from the Rnmt cKO vs. control CD4 T cell libraries were sequenced on a NextSeq 500 with a mid output v2.5 kit (150 cycles) in single end mode.
First the 10nt randomer barcode was extracted using umi_tools, then adapter sequences were trimmed from the reads using cutadapt (AGATCGGAAGAGCACACGTCTGAACTCCAGTCA). For paired end libraries only read one was used since reads were too short for the second read to be advantageous.
Reads were aligned to mm10 with gencode vM12 basic annotation using STAR 2.5.2b, then demultiplexed using umi_tools to remove PCR duplicates.
Secondary alignments and alignments with a MAPQ <20 were removed using samtools.
Bedgraph files were generated using deeptools, options -bs 10 --Offset 1 1 -of bedgraph.
Genome_build: Peaks were called using pureCLIP (Krakau et al., 2017) (options -iv 'chr1;chr2;chr3;chr4;chr5;' --mtc 10000 --ctr --dm 30) using the size matched input control for the input.
Genome_build: mm10 (GRCm38)
Supplementary_files_format_and_content: Bedgraph files have the counts of reads starting within the 10bp windows. Read start sites should be 1 nt downstream of crosslinked nucleotides so these footprints indicate areas of protein/RNA interaction in the LARP1 IP samples.
Supplementary_files_format_and_content: Bed files contain the coordinates of LARP1 binding peaks determined using PureCLIP.

source_name: naive CD4 T cells

growth_protocol: CD4 T cells were sorted from control and Rnmt cKO mouse lymph nodes using an EasySep mouse CD4 T cell isolation kit.

extract_protocol:
eCLIP was carried out essentially as described in the seCLIP protocol (Van Nostrand et al., 2017). Cells were thawed into lysis buffer (50 mM Tris-HCl pH 7.4, 100 mM NaCl, 1% NP-40 (Igepal CA630), 0.1% SDS, 0.5% sodium deoxycholate, 1:200 Protease Inhibitor Cocktail III (Sigma), 1:200 murine RNAse inhibitor (NEB)) and lysed on ice for 15 minutes. Lysates were incubated with 1µl/ml turbo DNase (Thermo Fisher Scientific) and 1µl/ml of a 1:2000 (1:1000 in test experiment) dilution of RNase1 (Thermo Fisher Scientific) in PBS for 5 mins at 37°C then returned to ice then centrifuged to remove insoluble material.
Lysates were precleared with protein G dynabeads (Thermo Fisher Scientific) at 4°C for 1hour. Lysates underwent immunoprecipitation with 50µl of protein G dynabeads preincubated with 4 µg of either LARP1, or isotype control antibody overnight at 4°C, 10% of the LARP1 samples were taken for the size matched input control. Beads were washed three times for 5 minutes with high salt wash buffer (50 mM Tris-HCl pH 7.4, 1 M NaCl, 1 mM EDTA, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate) with 2M urea. For the ProteinTech and Santa Cruz antibodies (and their isotype controls) used in the preliminary experiment a medium salt buffer (50 mM Tris-HCl pH 7.4, 600mM NaCl, 1 mM EDTA, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate) was used for these washes. RNA linker ligation was performed on bead using High concentration T4 RNA Ligase (NEB) for LARP1 and isotype libraries. RNA-protein complexes were denatured in NuPage loading buffer (Thermo Fisher Scientific) + 50mM DTT, resolved 3-8% Tris Acetate gel (Thermo Fisher Scientific) in Tris Acetate SDS buffer (Thermo Fisher Scientific), and transferred to a nitrocellulose membrane in NuPage transfer buffer (Thermo Fisher Scientific). RNA protein complexes and size matched input samples were cut from the membrane. RNA was released by incubating with Proteinase K for 20 minutes at 37°C, then with 420mg/ml urea in Proteinase K buffer for 20 minutes at 37°C and purified by phenol chloroform extraction. The RNA lnker was then ligated to the size matched input samples. All samples were reverse transcribed and ligated to a DNA linker ready for PCR amplification of libraries. Further details of the library preparation method are available in the seCLIP protocol (Van Nostrand et al., 2017).

treatment_protocol: CD4 T cells were suspended in PBS and exposed to 400mJ of 254nm UV then snap frozen on dry ice.

value: Naive CD4 T cells

tag: antibody

value: Rabbit IgG (Fisher 31235)


title: Rnmt_cKO_size_matched_input

description:
seCLIP library of size matched input RNA.
Rnmt cKO
ko_L1_pureclip_region.bed


title: Covid-19-Exo_3-26_89

value: TAACGTCGTATGCCGTCTTCTGCTTG


title: Rnmt_cKO_LARP1_IP

description:
seCLIP library of LARP1 interacting RNA.
Rnmt cKO
ko_L1_pureclip_region.bed

value: LARP1 (abcam ab86359)


title: Control_mouse_rabbit_IgG_IP

description:
seCLIP library of rabbit IgG control interacting RNA.
Control
con_L1_pureclip_region.bed


title: Control_mouse_size_matched_input

description:
seCLIP library of size matched control RNA.
Control
con_L1_pureclip_region.bed


title: Covid-19-Exo_3-26_90

value: In-house

value: TAATATCGTATGCCGTCTTCTGCTTG


title: Covid-19-Exo_3-26_82

value: TCTAGTCGTATGCCGTCTTCTGCTTG


title: Control_mouse_LARP1_IP

description:
seCLIP library of LARP1 interacting RNA.
Control
con_L1_pureclip_region.bed

value: Rnmt fl/fl CD4cre -


title: Covid-19-Exo_3-26_81

value: Mild

value: TCGCGTCGTATGCCGTCTTCTGCTTG


title: Rnmt_cKO_naive_CD4_3

description: Rnmt cKO 3

data_processing:
Libraries were sequenced on an Illumina NextSeq 500 using a high output 2 X 75bp kit.
Reads were aligned to mm10 with gencode vM12 basic annotation, and read counts for each gene calculated using STAR version 2.5.2b.
Gene expression was compared between controls and Rnmt cKO in R using EdgeR (Robinson et al., 2010), using a counts per million (CPM) threshold of 1 in at least three samples.
Genome_build: mm10 (GRCm38)
Supplementary_files_format_and_content: *ReadsPerGene.out.tab: Each file contains a tab-delimited table of reads per gene counted during alignment in STAR. The first column indicates the Ensembl gene id, the second column shows all alignments, the third shows forwards alignments, and the fourth shows reverse alignments (reverse aligned reads are the reads counts to use for this library strategy).
Supplementary_files_format_and_content: Rnmt_naive_CD4_edgeR.csv: The processed data file contains a comma-delimited table of the fold changes and statistical significance of comparisons between Rnmt cKO and control cells, and reads per million (RPM) of each gene calculated using EdgeR.

source_name: Naïve CD4 T cells

growth_protocol: Naïve CD4 T cells were FACS sorted (as: TCRb+, CD4+, CD62L+, CD44-, DAPI-) from lymph nodes.

extract_protocol:
RNA was prepared using Trizol.
Libraries were prepared at the Tayside Centre for Genomic Analysis with an Illumina TruSeq kit with Ribozero Gold rRNA depletion.

value: Male


title: Covid-19-Exo_3-26_80

value: TCGATTCGTATGCCGTCTTCTGCTTG


title: Rnmt_cKO_naive_CD4_2

description: Rnmt cKO 2


title: Covid-19-Exo_3-26_79

value: TCCTATCGTATGCCGTCTTCTGCTTG


title: Rnmt_cKO_naive_CD4_1

description: Rnmt cKO 1


title: Covid-19-Exo_3-26_78

value: TCCGTTCGTATGCCGTCTTCTGCTTG


title: Control_naive_CD4_2

description: Control 2


title: Covid-19-Exo_3-26_77

value: TCCACTCGTATGCCGTCTTCTGCTTG


title: BMDM_Ko_IFNγ_2h_rep2_RNA-seq [dataset 6]

city: Dallas

first: W. Lee

last: Kraus

state: TX

address: 5323 Harry Hines Blvd.

country: USA

institute: UT Southwestern Medical Center

description: RNA from Parp1 knockout IFNγ treated BMDM

data_processing:
The RNA-seq reads were aligned to the mm10 mouse reference genome using the Bowtie software package (Langmead et al, 2009) .
Mapped reads were further converted “wiggle” files and big wig files counting reads in non-overlapping 200-bp windows across the genome for presentation as genome browser tracks by using custom R scripts.
Genome_build: mm10
Supplementary_files_format_and_content: BigWig

growth_protocol: Bone marrow was harvested from 8-12 weeks old C57Bl6 mice and macrophages were differentiated in culture in DMEM supplemented with 20% FBS and LCCM.

extract_protocol:
Two replicates for the each of the different sets of BMDM cells were seeded at ~7.5 × 105 cells per well in 6-well plates and treated as described above.  The cells were collected and total RNA was isolated using the RNeasy kit (Qiagen) according to the manufacturer’s instructions.  The total RNA was then enriched for polyA+ RNA using Dynabeads Oligo(dT)25 (Invitrogen, 61002)
The polyA+ RNA was then used to generate strand-specific RNA-seq libraries as described previously (Zhong et al., 2011).  The RNA-seq libraries were subjected to QC analyses (i.e., number of PCR cycles required to amplify each library, the final library yield, and the size distribution of the final library DNA fragments) and sequenced using an Illumina HiSeq 2500 and NextSeq 500

treatment_protocol: BMDM were seeded in 6 well plates and treated with IFNγ (100 ng/mL)  for indicated times.

value: bone marrow derived macrophages (BMDM)

value: IFNγ


title: Covid-19-Exo_3-26_76

value: TCATCTCGTATGCCGTCTTCTGCTTG


title: Covid-19-Exo_3-26_84

value: Severe

value: TCTGATCGTATGCCGTCTTCTGCTTG


title: BMDM_Ko_IFNγ_1h_rep2_RNA-seq [dataset 6]

description: RNA from Parp1knockout IFNγ treated BMDM


title: Covid-19-Exo_3-26_83

value: TCTCCTCGTATGCCGTCTTCTGCTTG


title: U2AF complex PARCLIP (nuclear fraction)

city: Bethesda

first: Daniel

middle: R

last: Larson

address: 41 Medlar Dr.

institute: NIH

description: U2AF complex PARCLIP from nuclear fraction

data_processing:
Sequences were obtained using bcl2fastq and further processed through PARpipes (https://github.com/ohlerlab/PARpipe).
Pre-processing in the parpipe script was modified in the following order: Collapsing the sequences to obtain unique reads, trimming 4 Bases from the 5’ end, and the adapter sequence from the 3’ end using cutadapt.
Bowtie is implemented for alignment and paralyzer for cluster identification
The aligned.bam file was further filtered only for reads containing the characteristic TtoC mutation to provide IGV snapshots.
Genome_build: hg19
Supplementary_files_format_and_content: csv files, U2AF clusters,

source_name: HEK293 cells

growth_protocol: Cells were grown in DMEM containing 10% fetal bovine serum (Gibco), 100 U/ml penicillin, 100 µg/ml streptomycin, 10 µg/ml blasticidin (Gibco) and 100 µg/ml hygromycin B.

extract_protocol:
Cells were fractionated into nuclear and cytoplasmic fractions by resuspending in 3 mL mild hypotonic lysis buffer (10 mM TRIS-HCl pH 7.5, 50 mM NaCl, 3 mM MgCl2, 0.1% NP-40 and 10% glycerol) for 2 minutes and 5 minutes centrifugation at 400 g. The nuclear pellet was washed twice with 10 mL of the same buffer and lyzed with 1 mL of a RIPA buffer (150mM NaCl, 1% NP-40, 0.15 %DOX , 0.1 % SDS, 25 mM TRIS-HCl pH 7.5, Protease inhibitors, 1 U/μL RNAse T1 and 50 U/mL DNASE ) followed by sonication (3 X 10 sec; 60 % Amplitude on a VCX130 Vibra-Cell Ultrasonic Liquid Processor) and 30 minutes rotation at 4 oC. The lysate was cleared by centrifugation 10 minutes at 16000 G and diluted by addition of 2 volumes of 20mM TRIS-HCl pH 7.5. The FH-U2AF1/U2AF2 complex was Immunoprecipitated using the Anti-FLAG M2 Magnetic Beads (Sigma), treated with 1 U/mL RNase T1 (1/10000 Thermo Scientific RNase T1, EN0541) and washed 3 times with high salt wash buffer. To obtain the footprints of the U2AF complex 3’ pre-adenylated adapter (5'-rAppNNTCTGTGTGGAATTCTCGGGTGCCAAGG-L for the nuclear and rAppNNTGACTGTGGAATTCTCGGGTGCCAAGG-L for the Total cell ectract PAR-CLIP ) ligation was performed on beads using truncated T4 RNA Ligase 2 (NEB, M0242). The U2AF1/U2AF2-Crosslinked and 3’-adapter ligated footprints were obtained with proteinase K digestion on beads.
The recovered footprints were further size selected (19-35nt insert) by 15% PAGE -8M UREA using ligated 19-35 RNA oligos as markers. The size selected 3’ligeated footprint were ligated with a chimeric DNA -RNA 5’ adapter (GTTCAGAGTTCTACAGTCCGrArCrGrArUrCrNrNrNrN) and reverse transcribed using GCCTTGGCACCCGAGAATTCCA and Superscript IV reverse transcriptase following manufactures instructions (Invitrogen 18090010).

treatment_protocol: 20 15cm plates at 70% confluency were induced with Doxycycline for 24 hours followed by incubation with 4S-Uridine (300 μM) for 2 hours.

tag: transgene

tag: fraction

value: nuclear fraction


title: U2AF complex PARCLIP (whole cell)

description: U2AF complex PARCLIP from whole cell

value: whole cell


title: Primer extension sequencing (RAB7A)

description: primer extension from RAB7A 1st intron

data_processing:
Library strategy: primer extension sequencing
The adapter sequence from the 5’ and 3’ end was trimmed using cutadapt. Tophat is implemented for alignment.
Supplementary_files_format_and_content: junction.bed; junction reads count

source_name: Human Bronchial Epithelial Cells

growth_protocol: Human bronchial epithelial cells (HBECs) were grown in Keratinocyte-SFM medium (Gibco, 17005-042) with 1% penicillin-streptomycin (ThermoFisher, 15140122).

extract_protocol:
The metabolic labeling of newly synthesized RNA with 4thio-Uridine (4sU) and genome-wide RNA sequencing was performed as described by Bernd Radle et.al.. Briefly, for each sample, ~10^8 HBEC cells were incubated with 500 mM 4-thiouridine (Sigma, T4509) for 10 min. RNA was extracted with Trizol (ThermoFisher, 15596026).
To enrich the recursive splicing intermediates, we performed targeted reverse transcription followed by PCR amplification. First, ~2mg nascent RNA were purified with 4sU metabolic labeling. cDNA was synthesized with 50 ng nascent RNA as template per RT reaction with 16 RT-primers, individually. Next, we performed PCR amplification with Herculase II Fusion DNA polymerase through a touchdown PCR program (Agilent Technologies, 600675).

value: Human Bronchial Epithelial Cells


title: BMDM_Ko_IFNγ_1h_rep1_RNA-seq [dataset 6]


title: Covid-19-Exo_3-26_75

value: In-house

value: TCACTTCGTATGCCGTCTTCTGCTTG


title: Control_naive_CD4_3

description: Control 3

value: Rnmt fl/fl CD4cre -

value: Naive CD4 T cells


title: Sample_Input_F1

description:
total RNA
Sample_Input_F1

data_processing:
For all four nascent RNA-Seq samples, reads were trimmed for adapters and low-quality bases using Trimmomatic software. The Spliced Transcripts Alignment to a Reference (STAR) software was used to align trimmed reads to the reference genome hg19.
The splice junction output from STAR was then used for splice junction analysis.  For matching the splice junctions to known introns and exons, annotations from GenCode Release 19 were used. Exons were extracted directly from the annotations, while intron locations were computed using scripts provided with the RatchetScan program. Splice junctions supported by 2 or more uniquely mapping reads were compared against known annotations.
Supplementary_files_format_and_content: excel file, nascent RNA junction reads classification

source_name: input total RNA

extract_protocol:
The metabolic labeling of newly synthesized RNA with 4thio-Uridine (4sU) and genome-wide RNA sequencing was performed as described by Bernd Radle et.al.. Briefly, for each sample, ~10^8 HBEC cells were incubated with 500 mM 4-thiouridine (Sigma, T4509) for 10 min. RNA was extracted with Trizol (ThermoFisher, 15596026) and 10 ml total RNA was used as input control.
For genome-wide RNA sequencing, ribosomal RNA depletion (Illumina, Ribo-Zero rRNA removal Kit) was performed for both the 4sU labeled nascent RNA and total RNA. Libraries were generated with Trueseq Stranded Total RNA Sample preparation kits following the manufacturer’s instructions.


title: BMDM_Ko_Control_rep2_RNA-seq [dataset 6]

description: RNA from Parp1 knockout untreated BMDM

value: Untreated


title: BMDM_Ko_IFNγ_2h_rep1_RNA-seq [dataset 6]


title: Sample_Input_A2

description:
total RNA
Sample_Input_A2


title: BMDM_Ko_Control_rep1_RNA-seq [dataset 6]

description: RNA from Parp1 knockout untreated BMDM


title: Sample_Input_A1

description:
total RNA
Sample_Input_A1


title: BMDM_Wt_IFNγ_2h_rep2_RNA-seq [dataset 6]

description: RNA from Parp1 wildtype IFNγ treated BMDM


title: BMDM_Wt_IFNγ_2h_rep1_RNA-seq [dataset 6]


title: Sample_F2

description:
Nascent RNA
Sample_F2

source_name: Nascent RNA seq


title: BMDM_Wt_IFNγ_1h_rep2_RNA-seq [dataset 6]


title: Sample_F1

description:
Nascent RNA
Sample_F1


title: BMDM_Wt_IFNγ_1h_rep1_RNA-seq [dataset 6]


title: Sample_A2

description:
Nascent RNA
Sample_A2


title: BMDM_Wt_Control_rep2_RNA-seq [dataset 6]

description: RNA from Parp1 wildtype untreated BMDM


title: Sample_A1

description:
Nascent RNA
Sample_A1


title: Control_naive_CD4_1

description: Control 1

value: Male


title: Sample_Input_F2

description:
total RNA
Sample_Input_F2


title: BMDM_IFNγ_4h+PJ34_rep2_RNA-seq [dataset 5]

description: RNA from  IFNγ + PJ34 treated BMDM

source_name: BMDM_IFNγ_4h + PJ34

treatment_protocol: BMDM were seeded in 6 well plates and treated with IFNγ (100 ng/mL) for indicated times. For PARP inhibitor treatment condition, PJ34 (20 µM) was added 1 hour prior to the IFNγ treatment.

value: IFNγ + PJ34


title: BMDM_Wt_Control_rep1_RNA-seq [dataset 6]

description: RNA from Parp1 wildtype untreated BMDM


title: BMDM_IFNγ_4h+PJ34_rep1_RNA-seq [dataset 5]

description: RNA from  IFNγ + PJ34 treated BMDM


title: BMDM_IFNγ_2h+PJ34_rep2_RNA-seq [dataset 5]

description: RNA from  IFNγ + PJ34 treated BMDM

source_name: BMDM_IFNγ_2h + PJ34


title: BMDM_IFNγ_1h+PJ34_rep2_RNA-seq [dataset 5]

description: RNA from  IFNγ + PJ34 treated BMDM

source_name: BMDM_IFNγ_1h + PJ34


title: BMDM_IFNγ_1h+PJ34_rep1_RNA-seq [dataset 5]

description: RNA from  IFNγ + PJ34 treated BMDM


title: R878H_Ori_3

city: Tianjin

first: Jianwei

last: Wang

address: 10 Yuexin Street, Binhai New Area

country: China

institute: HAIHE LABORATORY OF CELL ECOSYSTEM

data_processing:
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence with Trim Galore (v0.6.6)
Trimmed reads were mapped to mouse reference genome GRCm38  using hisat2 (v2.2.0)
Reads count matrix were acquired with HTSeq (version 0.12.4)
Reads count matrix were normalized by using R package DESeq2 (1.22.0)
Genome_build: GRCm38
Supplementary_files_format_and_content: tab-delimited text files include normalized read counts for each Sample
Supplementary_files_format_and_content: Matrix table with raw gene counts for every gene and every sample

source_name: hematopoietic stem cells

growth_protocol: donor-derived hematopoietic stem cells were sorted from bone marrow transplanted recipient mice

extract_protocol:
Lysates were clarified from FACS-sorted  hematopoietic stem cells
First, 200 donor-derived HSCs (CD45.2+ Lineage- Sca-1+ c-kit+ CD34- CD150+) from oridonin-treated recipients were sorted into lysis buffer directly. The transcriptome libraries were prepared using Smart-seq2 method. Then, Agilent Bioanalyzer 2100 system (Agilent Technologies) was used to assess the insertion size, and HiSeq PE Cluster Kit v4-cBot-HS (Illumina) was used to perform the clustering of the index-coded samples according the manufacturer’s instructions. Finally, the libraries were sequenced using an Illumina Hiseq platform with 150-bp paired-end.

treatment_protocol: oridonin i.p treats recipient mice for 15 days

value: mouse-derived hematopoietic stem cells

tag: strain


title: WT_Ori_3

value: oridonin i.p treats recipient mice for 15 days


title: R878H_Ctl_4

value: vehicle


title: BMDM_PJ34_rep2_RNA-seq [dataset 5]

description: RNA from PJ34 treated BMDM

value: bone marrow derived macrophages (BMDM)


title: R878H_Ori_1


title: R878H_Ori_4


title: BMDM_IFNγ_2h+PJ34_rep1_RNA-seq [dataset 5]

description: RNA from  IFNγ + PJ34 treated BMDM


title: WT_Ori_2


title: WT_Ori_1


title: WT_Ctl_3


title: BMDM_IFNγ_4h_rep1_RNA-seq [dataset 5]

description: RNA from IFNγ treated BMDM

value: IFNγ


title: WT_Ctl_1


title: BMDM_IFNγ_2h_rep2_RNA-seq [dataset 5]

description: RNA from IFNγ treated BMDM


title: BMDM_IFNγ_2h_rep1_RNA-seq [dataset 5]

description: RNA from IFNγ treated BMDM


title: BMDM_IFNγ_1h_rep2_RNA-seq [dataset 5]

description: RNA from IFNγ treated BMDM


title: BMDM_IFNγ_1h_rep1_RNA-seq [dataset 5]

description: RNA from IFNγ treated BMDM


title: HSCs, 486 Veh

city: Indianapolis

first: Andrea

middle: Marie

last: Patterson

state: IN

address: 980 W. Walnut St.

department: Medicine

institute: Indiana University School of Medicine

data_processing:
FASTQ files were aligned with STAR v.2.5.2b using parameter “--outSAMmapqUnique 60”
Aligned reads in BAM files were counted with featureCounts from the subread package v.1.5.1 with parameters “-s 2 –p –Q 10”
Genome_build: mm10
Supplementary_files_format_and_content: tab-separated text file containing raw counts

source_name: Hematopoietic stem cells

growth_protocol: All cells direct ex vivo (taken from murine bone marrow immediately before treatment)

extract_protocol:
RNeasy Plus Mini Kit (Qiagen, Hilden, Germany)
Illumina Nextera XT

treatment_protocol: Murine bone marrow cells were treated with dmPGE2 or vehicle for 1 h at 37C, then immediately sorted/RNA-extracted

value: Vehicle

tag: age

value: Old

value: Hematopoietic stem cells


title: HSCs, 486 PGE


title: BMDM_Control_rep2_RNA-seq [dataset 5]

description: RNA from untreated BMDM

source_name: BMDM_Untreated control


title: HSCs, 485 PGE


title: HSCs, 485 Veh


title: R878H_Ctl_1


title: R878H_Ctl_2


title: BMDM_Control_rep1_RNA-seq [dataset 5]

description: RNA from untreated BMDM


title: HSCs, 484 PGE

value: Young


title: HSCs, 483 PGE


title: BMDM_IFNγ_4h_rep2_RNA-seq [dataset 5]

description: RNA from IFNγ treated BMDM


title: BMDM_PJ34_rep1_RNA-seq [dataset 5]

description: RNA from PJ34 treated BMDM


title: HSCs, 480 PGE


title: HSCs, 480 Veh


title: HSCs, 484 Veh


title: HSCs, 479 PGE


title: HSCs, 483 Veh


title: 13_Donor4_sgControl_NoStim

city: San Francisco

first: Zachary

last: Steinhart

state: CA

address: 1650 Owens St

department: Gladstone-UCSF Institute of Genomic Immunology

institute: J. David Gladstone Institutes

data_processing:
Adapters were trimmed from fastq files using cutadapt version 2.10 with default settings keeping a minimum read length of 20 bp.
Reads were mapped to the human genome GRCh38 keeping only uniquely mapping reads using STAR version 2.7.5b with the following settings “--outFilterMultimapNmax 1”.
Reads overlapping genes were then counted using featureCounts version 2.0.1 with the following settings “-s 2” and using the Gencode version 35 basic transcriptome annotation.
Genome_build: GRCh38
Supplementary_files_format_and_content: Matrix table with raw gene counts for every gene and every sample

source_name: Primary CD4+ T cells

growth_protocol: Primary human bulk T cells were cultured in X-VIVO 15 (Lonza Bioscience cat 04-418Q) supplemented with 5% FCS, 55mM 2-mercaptoethanol, 4mM N-Acetyl L-Cysteine, and 500 IU/ml recombinant human IL-2 (Amerisource Bergen cat 10101641). Primary T cell activation was done by anti-humanCD3/CD28 CTS dynabeads (Fisher Scientific cat 40203D) at a 1:1 cell to bead ratio at 1e6 cells/ml.

extract_protocol:
Cells were lysed and RNA was purified using Quick-RNA Microprep kit (Zymo Research) without the optional in-well DNase treatment step. Purified RNA was treated with TURBO DNase (Thermofisher Scientific) to remove potential contaminating DNA. RNA was subsequently purified using RNA Clean & Concentrator-5 kit (Zymo Research). RNA quality control was performed using an RNA ScreenTape assay (Agilent), with all samples having a RNA integrity number > 7.
RNA-seq libraries were prepared using the Illumina Stranded mRNA Prep kit, with 100ng of input RNA, following manufacturer's recommended protocol.

treatment_protocol: One day after activation, Bulk T cells were transduced with mCherry-2A-dCas9-VP64 lentivirus. Two days after activation, T cells were split into two populations and transduced with either a non-targeting control, or FOXQ1 transcriptional start site targeting sgRNA lentivirus. The following day 2ug/ml puromycin was added to the culture medium and cells were passaged every two days maintaining a minimum concentration of 0.3e6cells/ml. Eight days after initial activation, cells were sorted into CD4+mCherry+ populations and resumed culture in X-VIVO 15 without supplements. The next day cells were treated with 6.25ul/ml anti-CD3/CD28/CD2 Immunocult (Stim) or left untreated (NoStim). Cells were harvest 24 hours after stimulation.

value: Primary CD4+ T cells

tag: sgrna

value: No-Target Control


title: WT_Ctl_2


title: 15_Donor4_sgFOXQ1_NoStim

value: FOXQ1 Transcriptional Start Site


title: 16_Donor4_sgFOXQ1_Stim

value: anti-CD3/CD28/CD2 Immunocult


title: 12_Donor3_sgFOXQ1_Stim


title: HSCs, 479 Veh


title: 14_Donor4_sgControl_Stim


title: HSCs, 478 Veh


title: HSCs, 478 PGE


title: 11_Donor3_sgFOXQ1_NoStim


title: 10_Donor3_sgControl_Stim


title: 08_Donor2_sgFOXQ1_Stim


title: HSCs, 477 PGE


title: HSCs, 477 Veh


title: 07_Donor2_sgFOXQ1_NoStim


title: 06_Donor2_sgControl_Stim


title: H2AK119Ub_ChIP-seq_parental

city: Montreal

first: Jacek

last: Majewski

state: Quebec

address: 740 Dr Penfield Ave

department: Human Genetics

country: Canada

institute: McGill University

library_source: genomic

data_processing:
For ChIP-seq DNA input: Following alignment with BWA, bigwigs were generated with read coverage for 200-bp bins smoothed across 1-kb, computed with deepTools bamCoverage. Excludes mm10 ENCODE blacklist regions.
For ChIP-seq signal: Following alignment with BWA, bigwigs were generated with enrichment scores represented as input-normalized (log2-ratio), read-depth normalized signal per 200-bp bins, smoothed across 1-kb bins using deepTools. Excludes mm10 ENCODE blacklist regions.  See publication Methods for details.
For ChIP-seq signal peaks: Following alignment with BWA, SICER2 was used to call broad H2AK119Ub peaks. Processed output is a BED file in the format "chrom, start, end, read-count".
For RRBS: Following alignment with Bismark, bedGraph files were generated representing methylation scores at CpGs of at least 10X read coverage, computed using MethylDackel. See publication Methods for details.
Genome_build: mm10 for Mus musculus, dm6 for Drosophila melanogaster
Supplementary_files_format_and_content: Filetype suffix explanation: bw = bigWig

molecule: genomic DNA

source_name: C3H10T1/2 cells

growth_protocol: C3H10T1/2 cells were cultured in Dulbecco's modified Eagles' medium (DMEM, Invitrogen) with 10% fetal bovine serum (FBS, Sigma).

extract_protocol:
Cross-linking ChIP in MSCs was performed as described previously (Weinberg et al. 2019) using ~2x107 cells per immunoprecipitation. Prior to fixation, media was aspirated and cells washed once with PBS. Cells were cross-linked directly on the plate using 1% paraformaldehyde for 5 min at room temperature with gentle shaking. Glycine was added to quench (final concentration 125 mM, incubated for 5 min at room temperature), then cells were washed once with cold PBS, scraped off the plates, and pelleted. To obtain a soluble chromatin extract, cells were resuspended in 1 mL LB1 (50 mM HEPES, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100, 1x Complete protease inhibitor) and incubated rotating at 4°C for 10 min. Samples were centrifuged, resuspended in 1 mL LB2 (10 mM Tris-HCl pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1x Compete protease inhibitor), and incubated rotating at 4°C for 10 min. Finally, samples were centrifuged, resuspended in 1 mL LB3 (10 mM Tris-HCl pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% Na deoxycholate, 0.5% N-lauroylsarcosine, 1% Triton X-100, 1x Complete protease inhibitor), and homogenized by passing two times through a 27-gauge needle. Chromatin extracts were sonicated for 8 min (anti-HA ChIP) or 12 min (anti-histone PTM ChIP) using a Covaris E220 focused ultra-sonicator at peak power 140, duty factor 5, and cycles/burst 200. The lysates were incubated with 100 μl Pierce anti-HA beads (Themo Scientific, 88836) or 75 μl protein A Dynabeads (Invitrogen) bound to anti-H2AK119Ub (Cell Signaling, 8240) or anti-H3K27me3 (Cell Signaling Tech, 9733) antibodies and incubated overnight at 4°C with 5% kept as input DNA. Magnetic beads were sequentially washed with low salt buffer (150 mM NaCl; 0.1% SDS; 1% Triton X-100; 1 mM EDTA; 50 mM Tris-HCl), high salt buffer (500 mM NaCl; 0.1% SDS; 1% Triton X-100; 1 mM EDTA; 50 mM Tris-HCl), LiCl buffer (150 mM LiCl; 0.5% Na deoxycholate; 0.1% SDS; 1% Nonidet P-40; 1 mM EDTA; 50 mM Tris-HCl) and TE buffer (1 mM EDTA; 10 mM Tris-HCl). Beads were resuspended in elution buffer (1% SDS, 50 mM Tris-HCl pH 8.0, 10mM EDTA, 200 mM NaCl) and incubated for 30 min at 65°C. After centrifugation the eluate was reverse cross-linked overnight at 65°C. The eluate was then treated with RNaseA for 1 hr at 37°C and with Proteinase K (Roche) for 1 hr at 55°C and DNA was recovered using Qiagen PCR purification kit.
KAPA Hyper Prep kit

tag: chip antibody

value: H2AK119Ub (Cell Signaling Tech, #8240)

tag: cell line

value: C3H embryo-derived mesenchymal progenitor cells

value: Parental


title: 05_Donor2_sgControl_NoStim


title: 09_Donor3_sgControl_NoStim


title: 04_Donor1_sgFOXQ1_Stim


title: 03_Donor1_sgFOXQ1_NoStim


title: PBSCs, C09

data_processing:
FASTQ files were aligned with STAR v.2.5.2b using parameter “--outSAMmapqUnique 60”
Aligned reads in BAM files were counted with featureCounts from the subread package v.1.5.1 with parameters “-s 2 –p –Q 10”
Genome_build: hg19
Supplementary_files_format_and_content: tab-separated text file containing raw counts

source_name: Peripheral blood stem cells

growth_protocol: Direct ex vivo (cryopreserved) human apheresis product

treatment_protocol: Patients were treated with either Filgrastim alone or Meloxicam + Filgrastim for stem cell mobilization.

value: Filgrastim only

value: Female

tag: good vs poor mobilizer (cutoff 4e6 cd34+ cells/kg on first day apheresis)

value: Good

value: Peripheral blood stem cells


title: PBSCs, C07

value: Male

value: Poor


title: PBSCs, C06


title: PBSCs, C04


title: PBSCs, C05


title: PBSCs, C03


title: PBSCs, C02


title: PBSCs, C10


title: H2AK119Ub_ChIP-seq_parental_input

description: Used as input DNA for H2AK119Ub ChIP-seq in the parental background.


title: PBSCs, C01


title: PBSCs, C08


title: PBSCs, M25

value: Meloxicam + Filgrastim


title: PBSCs, M24


title: CRISPRi_DolcettoSetB_Donor2_IFNG_low

data_processing:
Fastq files were aligned to Calabrese (Addgene #92379, #92380) or Dolcetto (Addgene #92385, #92386) libraries using MAGeCK version 0.5.9.2 count command using --trim-5 22,23,24,25,26,28,29,30 argument
Genome_build: NA
Supplementary_files_format_and_content: Raw counts of sgRNAs

source_name: Primary CD4- T cells

extract_protocol:
After sorting, cells were washed with PBS, counted, pelleted, and resuspending at up to 5E6 cells per 400 µl of lysis buffer (1% SDS, 50 mM Tris, pH 8, 10 mM EDTA). The remaining protocol reflects additives/procedures performed per each 400 µl of sample. 16µl of NaCl (5M) was added, and the sample was incubated on a heat block overnight at 66°C. The next morning, 8µl of RNAse A (10mg/ml, resuspended in ddH2O) (Zymo, Cat #E1008) was added, and the sample was vortexed briefly, and incubated at 37°C for 1 hour. Next, 8µl of Proteinase K (20mg/ml) (Zymo, Cat #D3001) was added, the sample was vortexed briefly, and incubated at 55°C for 1 hour. A phase lock tube (Quantabio, Cat #2302820) was prepared for each sample by spinning down the gel to the bottom of the tube at 20,000g for 1 minute and then 400µl of Phenol:Chloroform:Isoamyl Alcohol (25:24:1) was added to each tube. 400µl of the sample was then added to the phase lock tube and the tube was shaken vigorously. The sample was centrifuged at maximum speed at room temperature for 5 minutes. The aqueous phase was transferred to a low-binding eppendorf tube (Eppendorf, Cat #022431021) and then 40µl of Sodium Acetate (3M), 1µl GlycoBlue (Invitrogen, Cat # AM9515), and 600µl of room temperature isopropanol was added. The sample was then vortexed and stored at -80°C for 30 minutes or until the sample had frozen solid. Next the sample was centrifuged at maximum speed at 4°C for 30 minutes, the pellet was washed with fresh 70% room temperature Ethanol, and allowed to air dry for 15 minutes. Pellets were then resuspended in Zymo DNA elution buffer (Zymo, Cat No: D3004-4-10), and placed on the heat block at 65°C for 1 hour to completely dissolve the genomic DNA.
sgRNA barcodes were amplified from genomic DNA using Takara Ex-Taq polymerase (Takara RR001C), using 40ug of isolated genomic dna as template per sample, following this protocol, https://media.addgene.org/cms/filer_public/61/16/611619f4-0926-4a07-b5c7-e286a8ecf7f5/broadgpp-sequencing-protocol.pdf. PCR amplicons were agarose gel purified using nucleoSpin Gel and PCR Clean‑up Mini kit (Machery-Nagel cat 740609.50)
CRISPR sgRNA amplicons

treatment_protocol: One day after activation, T cells were infected with dCas9-VP64 (CRISPRa) or dCas9-KRAB (CRISPRi) lentivirus. Two days after activation, T cells were split into two populations and infected with Calabrese Set A (addgene 92379) or Calabrese Set B (addgene 92380) lentivirus for CRISPRa, or Dolcetto Set A (addgene 92385) or Dolcetto Set B (addgene 92386) lentivirus for CRISPRi, each at an MOI of 0.5. The following day 2ug/ml puromycin was added to the culture medium and cells were passaged every two days maintaining a minimum concentration of 0.3e6cells/ml. Eight days after initial activation, cells were harvested, spun down and resuspended at 2e6 cells/ml X-VIVO 15 without supplements. Next day, cells were restimulated with anti-CD3/CD28/CD2 immunocult and treated with Brefeldin A for 9 hours, and fixed/stained for FACS. Cells were sorted into IL-2 low and high CD4+ T cells and IFN-γ low and high CD4- T cells

value: Primary CD4- T cells

tag: infection

tag: sorting


title: CRISPRi_DolcettoSetB_Donor2_IL2_low


title: CRISPRi_DolcettoSetB_Donor2_IL2_high


title: CRISPRi_DolcettoSetB_Donor2_IFNG_unsorted


title: CRISPRi_DolcettoSetA_Donor2_IFNG_unsorted


title: CRISPRi_DolcettoSetA_Donor2_IL2_high


title: CRISPRi_DolcettoSetA_Donor2_IL2_unsorted


title: CRISPRi_DolcettoSetA_Donor2_IL2_low


title: CRISPRi_DolcettoSetB_Donor2_IFNG_high


title: CRISPRi_DolcettoSetB_Donor2_IL2_unsorted


title: PBSCs, M23


title: CRISPRi_DolcettoSetA_Donor2_IFNG_high


title: 02_Donor1_sgControl_Stim


title: CRISPRi_DolcettoSetA_Donor2_IFNG_low


title: PBSCs, M20


title: 01_Donor1_sgControl_NoStim


title: PBSCs, M19


title: CRISPRi_DolcettoSetB_Donor1_IL2_unsorted


title: PBSCs, M17


title: CRISPRi_DolcettoSetB_Donor1_IFNG_unsorted


title: CRISPRi_DolcettoSetB_Donor1_IL2_high


title: CRISPRi_DolcettoSetB_Donor1_IL2_low


title: PBSCs, M26


title: PBSCs, M16


title: PBSCs, M15


title: CRISPRi_DolcettoSetB_Donor1_IFNG_high


title: PBSCs, M14


title: PBSCs, M21


title: CRISPRi_DolcettoSetA_Donor1_IL2_unsorted


title: PBSCs, M13


title: PBSCs, M11


title: PBSCs, M12


title: CRISPRi_DolcettoSetA_Donor1_IFNG_unsorted


title: CRISPRi_DolcettoSetA_Donor1_IL2_low


title: PBSCs, M09


title: PBSCs, M08


title: CRISPRi_DolcettoSetB_Donor1_IFNG_low


title: CRISPRi_DolcettoSetA_Donor1_IL2_high


title: CRISPRi_DolcettoSetA_Donor1_IFNG_high


title: PBSCs, M07


title: CRISPRi_DolcettoSetA_Donor1_IFNG_low


title: CRISPRa_CalabreseSetB_Donor2_IFNG_unsorted


title: PBSCs, M18


title: CRISPRa_CalabreseSetB_Donor2_IL2_unsorted


title: PBSCs, M03


title: CRISPRa_CalabreseSetB_Donor2_IL2_high


title: PBSCs, M01


title: PBSCs, M06


title: CRISPRa_CalabreseSetB_Donor2_IFNG_high


title: CRISPRa_CalabreseSetB_Donor2_IFNG_low


title: CRISPRa_CalabreseSetA_Donor2_IL2_unsorted


title: PBSCs, M10


title: donor_3_perifovea

city: Iowa City

first: Andrew

last: Voigt

state: Iowa

address: 375 Newton Road

institute: University of Iowa

data_processing:
FASTQ files were generated from the raw BCL files using Illumina’s bcl2fastq conversion program.
Reads were mapped to the human genome hg19 with STAR (version = 020201) and quantified with the featureCounts function from the Rsubread package (version = 1.28.1).
Differential expression analysis between foveal, perifoveal, and peripheral libraries was performed with edgeR (version = 3.26.8).
Genome_build: hg 19
Supplementary_files_format_and_content: Processed CPMs for all 12 samples are provided in a single supplemental spreadsheet

source_name: retina

extract_protocol:
Trephine punch biopsies were used to acquire foveal (1mm), perifoveal (4mm), and macular (8mm) retina. After dissection away from the RPE/choroid, neural retinal tissue was was flash frozen in liquid nitrogen. Total RNA was extracted with the Single Cell RNA Purification Kit (Norgen #51800)
Illumina TruSeq RNA Sample Preparation v2 library preparation kit (Illumina, San Diego, CA). Total RNA underwent 150 base pair paired-end sequencing on the Illumina HiSeq4000 platform (Illumina, San Diego CA).

tag: donor

tag: tissue

value: retina

tag: region

value: perifovea


title: donor_3_macula

value: macula


title: donor_4_perifovea


title: donor_2_perifovea


title: CRISPRa_CalabreseSetA_Donor2_IFNG_unsorted


title: donor_1_macula


title: donor_1_perifovea


title: donor_2_fovea

value: fovea


title: CRISPRa_CalabreseSetB_Donor2_IL2_low

value: Primary CD4+ T cells


title: donor_2_macula


title: CRISPRa_CalabreseSetA_Donor2_IFNG_high


title: CRISPRa_CalabreseSetA_Donor2_IFNG_low


title: donor_1_fovea


title: CRISPRa_CalabreseSetA_Donor2_IL2_high


title: CRISPRa_CalabreseSetB_Donor1_IL2_low


title: CRISPRa_CalabreseSetB_Donor1_IFNG_unsorted


title: CRISPRa_CalabreseSetB_Donor1_IL2_high


title: G164TP_3

city: London

first: Eleni

last: Maniati

address: John Vane Science Centre, Charterhouse Square

department: Barts Cancer Institute

institute: Queen Mary University of London

data_processing:
RNA-Seq reads were mapped to the human genome (hg19, Genome Reference Consortium GRCh37) using BWA mem (version 0.7.15-r1140) as part of the Oxford Genomics Centre pipeline.
Number of reads aligned to the exonic region of each gene were counted using htseq-count based on the Ensembl annotation.
Genes that achieved at least one read count per million reads (CPM) in at least 25% of the samples were kept. Conditional quantile normalisation (cqn) was performed counting for gene length and GC content and the log2 RPKM expression matrix was generated.
Genome_build: hg19 (GRCh37)
Supplementary_files_format_and_content: RPKM_cqn_MCMs.txt: Tab-delimited text file includes log2 RPKM values for each Sample.

source_name: adipocytes, mesothelial cells, fibroblasts, G164 HGSOC cells and platelets

growth_protocol: 14 days in suspension - DMEM:F12 + 10% FBS + 1% ITS + 1%P/S.

extract_protocol: RNA was prepared from cell cultures using the RNeasy Mini Kit and the RNase free DNase set (Qiagen).

treatment_protocol: 3D cultures were incubated with/without freshly isolated platelets in a 1:100 (cancer cells:platelets) ratio. 14 days later RNA was extracted from the gels according to manufacturer's protocol, starting with RLT buffer (350uL).

tag: culture type

value: 3D culture


title: donor_4_macula


title: CRISPRa_CalabreseSetB_Donor1_IFNG_high


title: CRISPRa_CalabreseSetB_Donor1_IFNG_low


title: donor_4_fovea


title: G164TP_2

value: adipocytes, mesothelial cells, fibroblasts, G164 HGSOC cells and platelets


title: G164TP_1


title: CRISPRa_CalabreseSetA_Donor1_IL2_unsorted


title: G164T_3

source_name: adipocytes, mesothelial cells, fibroblasts and G164 HGSOC cells


title: CRISPRa_CalabreseSetA_Donor1_IFNG_unsorted


title: CRISPRa_CalabreseSetA_Donor2_IL2_low


title: CRISPRa_CalabreseSetB_Donor1_IL2_unsorted


title: donor_3_fovea


title: MF_3

source_name: mesothelial cells plus fibroblasts

value: mesothelial cells plus fibroblasts


title: MF_2


title: G164T_2

value: adipocytes, mesothelial cells, fibroblasts and G164 HGSOC cells


title: CRISPRa_CalabreseSetA_Donor1_IFNG_high


title: CRISPRa_CalabreseSetA_Donor1_IFNG_low


title: CRISPRa_CalabreseSetA_Donor1_IL2_high


title: CRISPRa_CalabreseSetA_Donor1_IL2_low


title: Brain_3

city: Sherbrooke

first: Étienne

last: Fafard-Couture

state: Québec

address: 3201 Jean-Mignault

department: Biochemistry and functional genomics

institute: University of Sherbrooke

description:
Total RNA, ribodepleted
cDNA library generated with TGIRT

data_processing:
Trimming of paired-end reads was performed using Trimmomatic v0.36 (parameters: ILLUMINACLIP:<fastaWithAdaptersEtc>:2:12:10:8, TRAILING:30, LEADING:30, MINLEN:20) in order to remove adapters and low-quality reads.
FastQC v0.11.5 was used before and after trimming to assess the quality of the reads.
Alignment of reads to the reference human genome hg38 (v87) was performed using STAR v2.6.1a (parameters: --runMode alignReads, --outSAMunmapped None, --outSAMtype BAM SortedByCoordinates,   outFilterScoreMinOverLread 0.3, --outFilterMatchNminOverLread 0.3, --outFilterMultimapNmax 100, --winAnchorMultimapNmax 100, --alignEndsProtrude 5 ConcordantPair). The index needed to align reads was generated using STAR v2.6.1a (parameters: --runMode genomeGenerate, --sjdbOverhang 74), the reference human genome hg38 (v87) and the annotation file taken from ENSEMBL (v87) and supplemented with missing snoRNAs and tRNAs (available at https://zenodo.org/record/3981426/files/human_ensembl_87_wo_dup_v2_BB_v3.correct_annotation.gtf).
Gene expression normalized in count per million (CPM) and transcript per million (TPM) were obtained using CoCo v0.2.1p4 (parameters: cc, --countType both, --strand 1 --paired), a software optimizing the quantification of small non-coding RNAs nested in host gene.
Genome_build: hg38
Supplementary_files_format_and_content: Comma-separated value (.csv) file containing the id, name, biotype and abundance in each sample (in TPM) for all the genes detected in the TGIRT-Seq.

source_name: Brain tissue

extract_protocol:
Since RNA was directly purchased from BioChain for the different tissues, no extraction protocol was needed.
2 μg of DNA‑free total RNA was ribodepleted using Ribo-Zero Gold (Illumina) according to the manufacturer protocol. The resulting rRNA-free RNA was then purified with RNA Clean and Concentrator (RCC) kit (Zymo Research) using a modified protocol to retain RNA ≤ 80 nucleotides (400 μL ethanol 100 % per 50 μL sample). Purified RNA was fragmented 2-4 minutes (depending on the RNA Integrity Number) using NebNext Magnesium RNA Fragmentation Module (New England Biolabs) and once again purified with the RCC kit (Zymo Research) followed by dephosphorylation using T4 Polynucleotide Kinase (Epicentre) and final purification using, again, the RCC kit (Zymo Research). cDNAs were synthesized via TGIRT template-switching with 1 μM TGIRT-III reverse transcriptase (Ingex, LLC) for 15 min at 60℃, during which a DNA oligonucleotide containing the complement of an Illumina Read 2 sequencing primer-binding site became seamlessly linked to the 5’ cDNA end. After reaction cleanup, a 5’ adenylated DNA oligonucleotide containing the complement of an Illumina Read 1 sequencing primer-binding site was then ligated to the 3’ cDNA end with Thermostable 5’ AppDNA / RNA Ligase (New England Biolabs). Properly ligated cDNAs were amplified by PCR (12 cycles) to synthesize the second strand and add Illumina flowcell capture and index sequences. Libraries were purified with 2 rounds of Ampure XP beads (Beckman-Coulter) and evaluated on a 2100 Bioanalyzer (Agilent). Libraries were then pooled and sequenced on a NextSeq 500 platform (Illumina) (2 x 75 bp) using a NextSeq 500/550 High Output Kit v2.5 (150 cycles) (Illumina).
library strategy: TGIRT-Seq

value: Brain


title: CRISPRi_DolcettoSetA_Plasmid

molecule: other

organism: unidentified plasmid

source_name: plasmid

tag: sample type

value: plasmid


title: RNA-Seq SAMD1KO repl4

city: Marburg

first: Robert

last: Liefke

state: Hessen

address: Hans-Meerwein-Str. 2

department: Institute of Molecular Biology and Tumor Research (IMT)

country: Germany

institute: Philipps University of Marburg

data_processing:
ChIP-Seq data were aligned to mouse genome mm9 or mm10 using Bowtie 1.0, allowing one mismatch.
SAM files were converted to bigwig files using SamTools, and deepTools/bamCoverage. Data were normalized to RPKM.
RNA-Seq data were aligned to mouse transcriptome GenCode.M23 using RNA Star. Counts per gene were determined using FeatureCounts . Differentially expressed genes and normalized reads were determined using DeSeq2.
Genome_build: mm9, mm10
Supplementary_files_format_and_content: bw

growth_protocol: E14 mouse ES cells (E14TG2a) were cultured in DMEM, 15% FCS, 1 x L-Glutamine, 1 x Non-essential amino acids, 1 x Sodium pyruvate, 1 x Penicillin/Streptomycin , 0.15% β-mercaptoethanol and 100 Units/ml of LIF  on gelatin-coated plates.

extract_protocol:
ChIP experiments were performed in accordance to the One Day ChIP kit protocol (Diagenode). Total RNA was extracted by using the RNeasy Mini system (Qiagen) including an on-column DNaseI digestion.
For ChIP-Seq library five nanograms of precipitated DNA were used for indexed sequencing library preparation using the Microplex library preparation kit v2 (Diagenode). RNA-Seq libraries were prepared using the TruSeq Stranded mRNA Library Prep kit (Illumina).

tag: genotype


title: G164_SphPlat r3

description:
G164 high-grade serous ovarian cancer cell line with platelets.
RPKM_cqn_G164spheroids.txt

data_processing:
RNA-Seq reads were mapped to the human genome (hg19, Genome Reference Consortium GRCh37) using BWA mem (version 0.7.15-r1140) as part of the Oxford Genomics Centre pipeline.
Number of reads aligned to the exonic region of each gene were counted using htseq-count based on the Ensembl annotation.
Genes that achieved at least one read count per million reads (CPM) in at least 25% of the samples were kept. Conditional quantile normalisation (cqn) was performed counting for gene length and GC content and the log2 RPKM expression matrix was generated.
Genome_build: hg19 (GRCh37)
Supplementary_files_format_and_content: RPKM_cqn_G164spheroids.txt: Tab-delimited text file includes log2 RPKM values for each Sample.

source_name: G164 Spheroids + Platelets Donor 3

growth_protocol: DMEM F12, 1% P/S, 1% ITS, 2% B27, EGF 20 ng/mL, FGF 20 ng/mL.

extract_protocol:
RNA was prepared from cell cultures using the RNeasy Mini Kit and the RNase free DNase set (Qiagen).
Extracted RNA samples were sent to the Oxford Genomics Centre for processing. Library preps were made using RNA-Seq Ribozero kits (Illumina, Inc).

treatment_protocol: Cells (1x10^6) were incubated on low adherent flasks (T75) with platelets in a 1:250 (cell:platelets) ratio. 24 h later cells were extracted from the flasks, pelleted and washed (PBS). RNA was then extracted according to manufacturer's protocol (below), starting with RLT buffer (350uL).

tag: cell type

value: HGSOC cell line

tag: treatment

value: Platelets

value: Spheroids and platelets

tag: growth

value: 24h - low attachment flask. DMEM/F12 + 10% FBS


title: CRISPRi_DolcettoSetB_Plasmid


title: Brain_2


title: CRISPRa_CalabreseSetB_Plasmid


title: RNA-Seq SAMD1KO repl3

tag: antibody


title: RNA-Seq SAMD1KO repl2


title: G164_SphPlat r2

source_name: G164 Spheroids + Platelets Donor 2


title: Liver_3

source_name: Liver tissue

value: Liver


title: RNA-Seq SAMD1KO repl1


title: RNA-Seq WT repl4

value: WT


title: RNA-Seq WT repl3


title: Liver_2


title: G164_SphPlat r1

source_name: G164 Spheroids + Platelets Donor 1


title: G164T_1


title: MF_1


title: No TPA with BRAF [mRNA-Seq] 3

city: Salt Lake City

middle: L

last: Judson-Torres

state: Utah

address: 2000 Circle of Hope

department: Dermatology

institute: Huntsman Cancer Institute at the University of Utah

data_processing:
The human GRCh38 FASTA and GTF files were downloaded from Ensembl release 102.
The reference database was created using STAR version 2.7.2c with splice junctions optimized for 50 base pair reads.
Optical duplicates removed from NovaSeq runs via Clumpify v38.34
Reads were trimmed of adapters and aligned to the reference database using STAR in two pass mode to output a BAM file sorted by coordinates.
Mapped reads were assigned to annotated genes in the GTF file using featureCounts version 1.6.3
Non-coding features, histones, and ribosomal RNAs were removed and differentially expressed genes were identified using a 5% false discovery rate with DESeq2.
Genome_build: GRCh38
Supplementary_files_format_and_content: FeatureCounts output files combined into a single tab-delimited matrix

source_name: primary melanocytes

growth_protocol: Primary melanocytes were transduced with diBRAFV600E. They were grown in 254 + HMGS. Prior to experiment, HMGS2 samples (see characteristics) were switched to 254 + HMGS2 one passage prior to splitting for experiment.

extract_protocol:
Trizol, manufacture's protocol
Illumina TruSeq Stranded mRNA Library Prep (RIN 8-10) with UDI

treatment_protocol: Dox samples (see characteristics) received 15ng/uL doxycycline 4 days prior to harvesting RNA.

tag: background strain

value: HMGS2 + Dox


title: Adipo_1

source_name: adipocytes only

value: adipocytes only


title: G164_Sph r3

description:
G164 high-grade serous ovarian cancer cell line.
RPKM_cqn_G164spheroids.txt

source_name: G164 Spheroids

value: Untreated

value: Spheroids - monoculture


title: RNA-Seq WT repl2


title: CRISPRa_CalabreseSetA_Plasmid


title: No TPA with BRAF [mRNA-Seq] 2


title: No TPA with BRAF [mRNA-Seq] 1


title: Liver_1


title: RNA-Seq WT repl1


title: Skeletal_muscle_3

source_name: Skeletal muscle tissue

value: Skeletal muscle


title: G164_Sph r2


title: Brain_1


title: RNA-Seq L3MBTL3KO repl2

description:
NormalizedCounts-WT-KDM1AKO-L3MBTL3KO.txt
DeSeq2-KDM1AKOvsWT.txt
DeSeq2-L3MBTL3KOvsWT.txt

data_processing:
ChIP-Seq data were aligned to mouse genome mm9 or mm10 using Bowtie 1.0, allowing one mismatch.
SAM files were converted to bigwig files using SamTools, and deepTools/bamCoverage. Data were normalized to RPKM.
RNA-Seq data were aligned to mouse transcriptome GenCode.M23 using RNA Star. Counts per gene were determined using FeatureCounts . Differentially expressed genes and normalized reads were determined using DeSeq2.
Genome_build: mm9, mm10
Supplementary_files_format_and_content: txt

growth_protocol: E14 mouse ES cells (E14TG2a) were cultured in DMEM, 15% FCS, 1 x L-Glutamine, 1 x Non-essential amino acids, 1 x Sodium pyruvate, 1 x Penicillin/Streptomycin , 0.15% β-mercaptoethanol and 100 Units/ml of LIF on gelatin-coated plates.


title: Skeletal_muscle_2


title: G164_Sph r1


title: No TPA no BRAF [mRNA-Seq] 2


title: Skeletal_muscle_1


title: KDM1A_WT_repl1

value: KDM1A (Abcam, 17721)


title: RNA-Seq L3MBTL3KO repl3


title: RNA-Seq L3MBTL3KO repl1


title: No TPA no BRAF [mRNA-Seq] 1


title: Testis_3

source_name: Testis tissue

value: Testis


title: KDM1A_SAMD1KO_repl1


title: H3K27me3_WT

data_processing:
ChIP-Seq data were aligned to mouse genome mm9, mm10 or dm6 (spike-in) using Bowtie 1.0, allowing one mismatch.
SAM files were converted to bigwig files using SamTools, and deepTools/bamCoverage. Data were normalized to RPKM and spike-in control.
Genome_build: mm9, mm10, dm6
Supplementary_files_format_and_content: bw


title: Testis_2


title: RNA-Seq KDM1AKO repl3


title: TPA with BRAF [mRNA-Seq] 3

value: HMGS + Dox


title: TPA with BRAF [mRNA-Seq] 2


title: Testis_1


title: RNA-Seq KDM1AKO repl2


title: H3K4me3_WT_repl2

value: H3K4me3 (Diagenode, C15410003)


title: H3K4me3_SAMD1KO_repl2


title: TPA with BRAF [mRNA-Seq] 1


title: No TPA no BRAF [mRNA-Seq] 3


title: RNA-Seq KDM1AKO repl1


title: RNA-Seq WT2 repl3


title: H3K27me3_SAMD1KO


title: TPA no BRAF [mRNA-Seq] 3

value: HMGS


title: H3K4me2_WT_repl2

value: H3K4me2 (Diagenode, C15410035)


title: TC71 STAG2 KO sg4 rep1

city: Cambridge

first: Gabriela

last: Alexe

state: MA

address: 415 Main St.

department: Computational Biology and Bioinformatics

institute: Broad Institute

data_processing:
The CRISPR plasmids pSpCas9(BB)-2A-GFP (PX458) and lentiCRISPR v2 were acquired from Addgene. STAG2 wild-type clones and and STAG2 null clones by CRISPR Cas9 editing were created on  A673 cells (STAG2 wild-type: A673.sgNT-1c4, A673.sgNT-2c3, A673.sgLacZ-2c1; STAG2 null: A673.sgSTAG2-1c4, A673.sgSTAG2-1c6, A673.sgSTAG2-4c3, A673.sgSTAG2-4c5)  and on TC71 cells (STAG2 wild-type:  TC71.sgNT-1c6, TC71.sgNT-2c5; STAG2 null: TC71.sgSTAG2-1c6, TC71.sgSTAG2-4c15, TC71.sgSTAG2-4c18, TC71.sgSTAG2-1c3). Total RNA was extracted with an RNeasy Kit (Qiagen) from STAG2 wild-type and STAG2 null clones (A673.sgNT-1c4, A673.sgNT-2c3, A673.sgLacZ-2c1, A673.sgSTAG2-1c4, A673.sgSTAG2-1c6, A673.sgSTAG2-4c3, A673.sgSTAG2-4c5, TC71.sgNT-1c6, TC71.sgNT-2c5, TC71.sgSTAG2-1c6, TC71.sgSTAG2-4c15, TC71.sgSTAG2-4c18, TC71.sgSTAG2-1c3). For the A673 clones, Poly(A) RNA was isolated using the NEBNext mRNA magnetic isolated module (New England Biolabs) and paired-end libraries were prepared using the NEBNext Ultra Directional RNA Library Prep Kit for Illumina (New Englad Biolabs) according to the manufacturer’s protocols with the following modifications: the PCR library enrichment conditions were adjusted to 12 cycles and the PCR library reaction was purified twice by size selection with Agencourt AMPure XP beads (Beckman Coulter). Libraries were subjected to 50 base paired-end sequencing (Illumina HiSeq 2000).    Total RNA was extracted from triplicate samples of TC71 STAG2 clones with the RNeasy Kit and on-column DNA digestion (Qiagen). Poly(A) mRNA was isolated and libraries were prepared using the TruSeq Stranded mRNA Kit (Illumina) according to the manufacturer’s protocol.  Strand-specific mRNA seq libraries were pooled and sequenced on a NextSeq500 instrument with single-end 75bp reads to a depth of 30-40M reads/sample.
Quality control tests for the sequenced reads  were performed using the FASTQC v0.11.5 software (www.bioinformatics.babraham.ac.uk/projects/fastqc/). The reads were aligned to the GRCh37/hg19 human genes by using Tophat2 v2.1.1. Quality control tests for the aligned reads and for the replicate consistency were performed by using the qualimap v2.2.1 and the SARTools  pipelines.
Gene level reads and gene level expression estimated as log2(1+FPKM) (Fragments Per Kilobase of transcript per Million mapped reads) scores were computed using the Feature Counts method implemented in the Bioconductor v3.5  RSubread v1.5.3 package.
Genome_build: GRCh37/hg19
Supplementary_files_format_and_content: Tab-delimited text file include gene level counts values for each sample.

source_name: TC71 Ewing sarcoma cell line

extract_protocol:
RNAseq library construction protocol - A673 cells:  For the A673 clones, Poly(A) RNA was isolated using the NEBNext mRNA magnetic isolated module (New England Biolabs) and paired-end libraries were prepared using the NEBNext Ultra Directional RNA Library Prep Kit for Illumina (New Englad Biolabs) according to the manufacturer’s protocols with the following modifications: the PCR library enrichment conditions were adjusted to 12 cycles and the PCR library reaction was purified twice by size selection with Agencourt AMPure XP beads (Beckman Coulter). Libraries were subjected to 50 base paired-end sequencing (Illumina HiSeq 2000).
RNAseq library construction protocol - TC71 cells:  Poly(A) mRNA was isolated and libraries were prepared using the TruSeq Stranded mRNA Kit (Illumina) according to the manufacturer’s protocol.  Strand-specific mRNA seq libraries were pooled and sequenced on a NextSeq500 instrument with single-end 75bp reads to a depth of 30-40M reads/sample.

tag: genotype/variaton

value: STAG2 null

tag: crispr-cas9 guides


title: TPA no BRAF [mRNA-Seq] 2


title: RNA-Seq WT2 repl2

tag: cell line


title: TC71 STAG2 KO sg4 rep2


title: TC71 STAG2 KO sg1 rep2


title: TC71 STAG2 KO sg1 rep1


title: TPA no BRAF [mRNA-Seq] 1


title: RNA-Seq WT2 repl1


title: No TPA with BRAF [miRNA-Seq] 3

data_processing:
The human GRCh38 FASTA and GTF files were downloaded from Ensembl release 102.
The reference database was created using STAR version 2.7.2c with splice junctions optimized for 50 base pair reads.
Reads were trimmed of adapters and aligned to the reference database using STAR in two pass mode to output a BAM file sorted by coordinates.
Mapped reads were assigned to annotated genes in the GTF file using featureCounts version 1.6.3
UMI tags were extracted and PCR duplicates removed with custom perl script
Features were counted with Subread 1.6.3
Genome_build: GRCh38
Supplementary_files_format_and_content: counts output files combined into a single tab-delimited matrix

extract_protocol:
Trizol, manufacture's protocol
Qiagen Qiaseq miRNA Library Prep with UDI


title: IgG_SAMD1KO

value: IgG (Diagenode, C15410206)


title: RNA-Seq Diff-d7 SAMD1KO repl3

description:
NormalizedCounts-Differentiation.txt
DeSeq2-Differentiation-SAMD1KOvsWT.txt

tag: time

value: Differentiation day 7


title: No TPA with BRAF [miRNA-Seq] 2


title: RNA-Seq Diff-d7 SAMD1KO repl2


title: IgG_WT


title: TC71 STAG2 WT  rep1

value: STAG2 wild-type


title: SAMD1_SAMD1KO_repl1

value: SAMD1 (self-made)


title: No TPA with BRAF [miRNA-Seq] 1


title: H3K4me2_SAMD1KO_repl2


title: DNMT3A1-K299I_RRBS_sgRing1a-b

source_name: C3H10T1/2 cells

extract_protocol:
DNA was extracted from flash frozen cell pelets of about 2 million cells using DNeasy Blood & Tissue Kit (Qiagen, 69509)
100 ng of genomic DNA were digested with 100U of MspI (NEB) and end-repaired/A-tailed using Kapa Hyper Prep kit (Kapa Biosystems). After ligation of Illumina-sequencing compatible indexes, DNA was purified using a 1X Agencourt AMPure XP bead clean up (Beckman Coulter). Bisulfite conversion was carried out using the Zymo EZ DNA kit (Zymo Research) using the following program: 55 cycles: 95°C 30s, 50°C 15 min, 4°C hold. Libraries were amplified 17 cycles using Uracil+ Ready mix (Kapa Biosystems, KK2801). The resulting libraries, mean size 388bp, were normalized to 3nM, pooled and clustered on a pair end read flow cell and sequenced for 100 cycles on a NovaSeq6000 (Illumina).

value: C3H embryo-derived mesenchymal progenitor cells

value: sgRing1a/b line expressing FLAG-HA tagged K299I DNMT3A1


title: RNA-Seq Diff-d7 SAMD1KO repl1


title: RNA-Seq Diff-d7 WT repl3

value: WT


title: No TPA no BRAF [miRNA-Seq] 3


title: No TPA no BRAF [miRNA-Seq] 2


title: SAMD1_WT_repl1


title: DNMT3A1-W330R_RRBS_sgRing1a-b

value: sgRing1a/b line expressing FLAG-HA tagged W330R DNMT3A1


title: RNA-Seq Diff-d7 WT repl2


title: TC71 STAG2 WT rep2


title: RNA-Seq Diff-d7 WT repl1


title: No TPA no BRAF [miRNA-Seq] 1


title: DNMT3A1-K299I_RRBS_parental

value: Parental line expressing FLAG-HA tagged K299I DNMT3A1


title: DNMT3A1-WT_RRBS_sgRing1a-b

value: sgRing1a/b line expressing FLAG-HA tagged wild-type DNMT3A1


title: TPA with BRAF [miRNA-Seq] 3


title: RNA-Seq Diff-d0 SAMD1KO repl3

value: Differentiation day 0


title: DNMT3A1-W330R_RRBS_parental

value: Parental line expressing FLAG-HA tagged W330R DNMT3A1


title: TPA with BRAF [miRNA-Seq] 2

value: HMGS + Dox


title: HFD_caloric_restriction_rep5

type: RNA

city: Boston

first: Grace

last: Daher

address: 1 Joslin Place

institute: Joslin Diabetes Center

hyb_protocol: Approximately 3.5 ug cDNA was hybridized to GeneChip Mouse Gene 2.0 ST arrays for 16 hours at 45°C. Arrays were washed and stained using Affymetrix Fluidics Station 450 with reagents from Affymetrix.

scan_protocol: GeneChips were scanned using the GeneChip Scanner 3000 from Affymetrix.

data_processing: The data were normalized on Expression Console using RMA.

label: biotin

source_name: hepatic tissue

label_protocol: Fragmented and labeled cDNA were prepared according to the WT Plus Affymetrix protocol from 250 ng total RNA.

growth_protocol: 6 weeks old male C57Bl/6J were treated with 60% high fat diet (HFD) ad libitum for 4 weeks to induced obesity

extract_protocol: Total RNA was isolated from mice treated for 4 weeks in each of the 3 experimental groups (n=3-5 per group) using TRIzol (Life Technologies) and choloroform. RNA pellet were washed with propanol and 75% ethanol.

treatment_protocol: Mice were treated with either (1) Continued HFD for additional 4 weeks (2) HFD +canagliflozin (25-30mg/kg) (3) Weight match control to group(2) with 15-30% caloric restriction.

tag: strain

value: caloric restriction


title: RNA-Seq Diff-d0 SAMD1KO repl2


title: DNMT3A1-WT_RRBS_parental

value: Parental line expressing FLAG-HA tagged wild-type DNMT3A1


title: RNA-Seq Diff-d0 SAMD1KO repl1


title: TPA with BRAF [miRNA-Seq] 1


title: DNMT3A1-D333N_ChIP-seq_parental-rep-2_input

tag: chip antibody

value: Parental line expressing FLAG-HA tagged DNMT3A1 D333N


title: RNA-Seq Diff-d0 WT repl3


title: TPA no BRAF [miRNA-Seq] 3

value: HMGS


title: TPA no BRAF [miRNA-Seq] 2


title: RNA-Seq Diff-d0 WT repl2


title: DNMT3A1-D333N_ChIP-seq_parental-rep-2

value: HA (ThermoFisher, #88836)


title: HFD_caloric_restriction_rep4


title: DNMT3A1-W330R_ChIP-seq_parental-rep-2_input

value: Parental line expressing FLAG-HA tagged DNMT3A1 W330R


title: TPA no BRAF [miRNA-Seq] 1


title: RNA-Seq Diff-d0 WT repl1


title: DNMT3A1-W330R_ChIP-seq_parental-rep-2


title: HFD_CANA_rep4

value: canagliflozin (30mg/kg)


title: HFD_rep4

value: control


title: HFD_rep3


title: HFD_CANA_rep5


title: L3MBTL3(CT)_SAMD1KO

value: L3MBTL3-CT (self-made)


title: HFD_caloric_restriction_rep3


title: HFD_caloric_restriction_rep2


title: HFD_rep2


title: HFD_CANA_rep2


title: DNMT3A1-R318W_ChIP-seq_parental-rep-2_input

value: C3H embryo-derived mesenchymal progenitor cells

value: Parental line expressing FLAG-HA tagged DNMT3A1 R318W


title: Human CD19- antibody-secreting cells, AFRS

city: Pittsburgh

first: Richard

middle: Paul

last: Ramonell

state: PA

address: 5859 Phillips Ave

department: Department of Medicine

institute: University of Pittsburgh

description: AFRS_1_cd19pos_cd19neg_ighv_allisotypes.csv

data_processing:
Library strategy: VDJ-seq
Illumina Casava 1.8.2 software used for basecalling
Sequenced reads were demultiplexed, filtered, and analyzed using Cell Ranger 3.1.0 software
Fasta files were aligned to IMGT using HighV-QUEST tool 1.8.3
Genome_build: IMGT
Supplementary_files_format_and_content: comma separated values files include filtered sequence data, metadata from each sample, and secondary analysis of repertoire characteristics (Vh region mutation frequency)

source_name: CD19- ASCs, donor 1

extract_protocol:
ASCs were sorted into R5 (RPMI + 5% FBS + antibiotic/antimycotic) and then loaded onto a 10X Genomics Next GEM Chromium chip.
scRNA libraries were prepared for sequencing using standard 10X Genomics and Illumina protocols

treatment_protocol: Following polypectomy, ASCs were isolated from fresh nasal polyp tissue using enzymatic (Liberase TL) and mechanical dissociation. ASCs were stained and FACS sorted using a gating strategy as follows: CD19+CD27hiCD38hi (CD19+ ASCs) or CD19-CD38+CD138+ (CD19- ASCs).

value: Nasal-polyp derived ASCs

tag: scrna seq platform

value: 10X Genomics

tag: cell marker


title: Human CD19+ antibody-secreting cells, AERD

description: AERD_cd19pos_cd19neg_ighv_allisotypes.csv

source_name: CD19+ ASCs, donor 2


title: DNMT3A1-R318W_ChIP-seq_parental-rep-2


title: DNMT3A1-K299I_ChIP-seq_parental-rep-2_input

value: Parental line expressing FLAG-HA tagged DNMT3A1 K299I


title: L3MBTL3(NT)_SAMD1KO

value: L3MBTL3-NT (self-made)


title: L3MBTL3(CT)_WT

value: WT


title: HFD_CANA_rep3


title: Human CD19+ antibody-secreting cells, AFRS

source_name: CD19+ ASCs, donor 1


title: HFD_caloric_restriction_rep1


title: L3MBTL3(NT)_WT


title: HFD_rep1


title: Human CD19- antibody-secreting cells, AERD

source_name: CD19- ASCs, donor 2


title: H3K27me3 on A673 STAG2 KO [BA_A673_STAG2_sg1KO_H3K27me3]

data_processing:
Quality control tests for unmapped sequences were performed based on the FastQC v.0.11.5 software (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). The H3K27me3 and the H3K4me3 ChIP-Seq data sets were aligned to the GRCh37/hg19 human genes PCR duplicates were removed with the Picard v2.18.2 MarkDuplicates tool (Li and Durbin, 2010). The H3K27me3 data was spiked-in with the Active Motif Drosophila Melanogaster kit and mapped to the Drosophila Melanogaster dm6 genes using bowtie2-2.3.5 with the standard options. The Active Motif Spike-in Normalization protocol was then applied to the H3K27me3  hg19 sample by multiplying the human tag counts with the normalization factors derived from the uniquely mapped Drosophila reads as ratios between the sample with the lowest dm6 counts vs. the dm6 counts for that sample. For the  H3K4me3 mark  the reads mapped on the STAG2 wild-type clones A673.sgNT-1c4 and A673.sgNT-2c3 were merged and labeled as "STAG2 WT". For the H3K27me3 mark  the reads mapped on the STAG2 knockout clone A673.sgSTAG2-1c6 were labeled as "STAG2 sg1KO".  The mapped reads for individual and merged clones were normalized in units of Reads Per Kilobase per Million (RPKM or rpm/bp) and coverage tracks for the RPKM signal were created as bigwig files for bins of size 20 base pairs by using the bamCoverage tool available in deepTools v2.5.3. (Ramirez et al., 2016).
Peak calling was performed against input control using the model-based MACS2 v2.1.1.20160309 software (Zhang et al., 2008), with the cut-off FDR ≤ 0.01. Narrow peaks were identified for all marks except H3K27Me3 for which macs2 was called with the broad peak option.  Area under Curve (AUC) RPKM normalized signal across genomic regions was computed with the bwtool software (Pohl and Beato, 2014). Peaks with low area under curve coverage (< 300 RPKM)  were disregarded and the ENCODE black-listed regions for hg19 (available at https://www.encodeproject.org/annotations/ENCSR636HFF/) were removed from each set of peak regions. Quality control tests for the mapped reads were performed by using the ChIPQC library available from Bioconductor v3.6 (Carroll et al., 2014). The distances between replicates for STAG2 WT and STAG2 KO clones were estimated based on the multiBamSummary function available from the deepTools v2.5.3. (Ramirez et al., 2016) and visualized on correlation heatmaps and PCA plots. The peaks were annotated with the closest hg19 genes by using the annotatePeaks function available in the Homer v4.11 package (Heinz et al., 2010) and the GREAT annotation platform (McLean et al., 2010).
Genome_build: GRCh37/hg19
Supplementary_files_format_and_content: Bigwig files for normalized coverage signal in units of reads per million mapped reads per bp (rpm/bp).  Coverage was computed for  bins of size 20 base pairs by using the bamCoverage module available in deepTools v2.5.3.  The Active Motif Spike-in Normalization protocol was then applied to each hg19 sample -except Input- by multiplying the human tag counts with the normalization factors derived from the uniquely mapped Drosophila reads as ratios between the sample with the lowest dm6 counts vs. the dm6 counts for that sample.

source_name: A673 clone A673.sgSTAG2-1c6 with STAG2 knocked-out

extract_protocol:
Antibodies:   Antibodies: Rabbit anti-H3K27me3 Cell Signaling Cat# 4395, RRID:  AB_11220433; H3K4me3:  Abcam, ab8580.
Cells (20 million per ChIP reaction , which required 40 million cells) were crosslinked with warm 1% methanol-free formaldehyde (ThermoFisher) for 10 minutes at room temperature rotating at 12 RPM. The reaction was quenched by adding glycine to a final concentration of 0.125M and incubating for an additional 5 minutes at room temperature rotating at 12 RPM. Cell pellets were washed three times with ice cold PBS and resuspended in 1 ml of SDS lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl pH8) supplemented with protease inhibitor cocktail including phenylmethylsulfonyl fluoride (PMSF) and incubated at RT for 2 minutes with gentle rotation. Lysates were centrifuges at 15,000 G for 10 minutes at 4°C and the pellet was re-suspended in 900 µl of ChIP IP buffer (2:1 SDS lysis buffer : triton dilution buffer), transferred to milliTUBE (covaris). Sonication was performed on a E220 Focus Ultra Sonicator (Covaris) using the setting (duty cycle 5%, peak power 140W, cycles per burst 200, Temperature 4°C, time 30 minutes/millitube). ChIP inputs from sheared chromatin were de-crosslinked by adding de-crosslinking buffer (NaHCO3, NaCl, RNase A, Proteinase K) and incubating for two hours at 65°C in a thermal cycler. The remaining sheared chromatin was incubated with primary antibody coupled to Protein A DynaBead (Beckman Coulter, antibody bead conjugation was performed for 16 hours) overnight, rotating at 4°C. As a calibration control, antibody against a drosophila specific histone variant H2Av and drosophila chromatin (active motif) were used as per the recommendation of the manufacturer.  The next day, ChIP product was eluted from the Dynabeads in 100 µl of elusion buffer and de-crosslinked for 12 hours at 65°C. For both Input and chipped material, AMPure XP beads (Beckman coulter) were used to purify DNA.
Chromatin immunoprecipitation sequencing for  H3K27me3, H3K4me3:  ChIP-seq libraries were prepared using Swift S2 Acel reagents on a Beckman Coulter Biomek i7 liquid handling platform from approximately 1 ng of DNA according to the manufacturer’s protocol and 14 cycles of PCR amplification. Finished sequencing libraries were quantified by a Qubit fluorometer and samples were QC’D using a Bioanalyzer Tapestation (Agilent Technologies 2200) to determine  fragment size. Library pooling and indexing was evaluated with shallow sequencing on an Illumina MiSeq. Subsequently, libraries were sequenced on a NovaSeq targeting 40 million 100bp read pairs by the Molecular Biology Core facilities at Dana-Farber Cancer Institute.

value: A673 Ewing sarcoma

value: STAG2 knocked-out


title: DNMT3A1-delPWWP_ChIP-seq_parental-rep-2_input

value: Parental line expressing FLAG-HA tagged DNMT3A1 delPWWP


title: TdgWT-Th2-1st-PB-rep1

city: Chiba

first: Atsushi

last: Onodera

address: Chuo-ku Inohana 1-8-1

department: Immunology

country: Japan

institute: Chiba University

data_processing:
Library strategy: PB-seq
The BS and PB reads were mapped to mouse genome mm9 using the BSMAP mapping tool. The mapping was done using the paired end datasets with the following parameter values: -p 4 -w 2 -v 5 -q 30 for BS-seq, and -p 4 -w 2 -v 0 -q 30 for PB-seq.
For each of the samples, the number of reads in which each cytosine within the amplicons was converted into thymine was counted.
For BS-seq, these counts were used to calculate the proportions for each cytosine to be nonmethylated/formylmethylated/carboxylmethylated (T), or methylated/hydroxymethylated (C).
For PB-seq, to compensate redundant reads derived from the opposite strand, number of reads standing for nonmethylated/methylated/hydroxymethylated (C) was multiplied by 0.5, and the proportions for each cytosine to be formylmethylated/carboxylmethylated (T) was calculated.
Genome_build: mm9
Supplementary_files_format_and_content: methylation_ratio .txt file

source_name: Th2-1st cells

growth_protocol: For Th2 polarization naïve CD4+ T cells were stimulated with plate-bound anti-CD3 (clone 2C11) and anti-CD28 (clone 37.51) antibodies at 1 μg/ml in the presence of 1000 U/ ml IL-4, 1 μg/ml anti-IFN-γ, for 2 days. On day 3 of culture, cells were removed from TCR/CD28 costimulation conditions and expanded in the presence of 20 U/mL IL-2 for 3 days.

extract_protocol:
Genomic DNA (gDNA) was isolated by PureLink Genomic DNA Mini Kit (Thermo Fisher Scientific). 0.5 μg of gDNA samples were treated with sodium BS (MethylCode Bisulfite Conversion kit; Invitrogen).
The PCR amplicons were generated using the PyroMark PCR kit (QIAGEN) and quantified using Qubit assays (Invitrogen). PCR amplicons were pooled and then used for library preparation using NEB Next DNA Library Modules for Illumina platform (New England Biolabs, Inc.).
bisulfite or pyridine borane sequencing

value: wild type

value: T-cells from the spleen and lymphnodes


title: DNMT3A1-K299I_ChIP-seq_parental-rep-2

data_processing:
For ChIP-seq DNA input: Following alignment with BWA, bigwigs were generated with read coverage for 200-bp bins smoothed across 1-kb, computed with deepTools bamCoverage. Excludes mm10 ENCODE blacklist regions.
For ChIP-seq signal: Following alignment with BWA, bigwigs were generated with enrichment scores represented as input-normalized (log2-ratio), read-depth normalized signal per 200-bp bins, smoothed across 1-kb bins using deepTools. Excludes mm10 ENCODE blacklist regions.  See publication Methods for details.
For ChIP-seq signal peaks: Following alignment with BWA, SICER2 was used to call broad H2AK119Ub peaks. Processed output is a BED file in the format "chrom, start, end, read-count".
For RRBS: Following alignment with Bismark, bedGraph files were generated representing methylation scores at CpGs of at least 10X read coverage, computed using MethylDackel. See publication Methods for details.
Genome_build: mm10 for Mus musculus, dm6 for Drosophila melanogaster
Supplementary_files_format_and_content: Filetype suffix explanation: bw = bigWig

value: HA (ThermoFisher, #88836)


title: H3K4me3 on A673 STAG2 WT [A673_STAG2WT_H3K4me3]

source_name: A673 clones A673.sgNT-1c4 and A673.sgNT-2c3 with STAG2 WT

value: STAG2 wild-type


title: DNMT3A1-delPWWP_ChIP-seq_parental-rep-2


title: MM330_siNT_E2_3

city: Aurora

last: Goodspeed

state: CO

address: 12801 E 17th Ave, Mail Stop 8303

institute: University of Colorado Anschutz Medical Campus

description:
si_treatment: siNT_E2
Compiled_counts.txt

data_processing:
Illumina adapters were removed, along with the first 12 base pairs using BBDuk and reads <50bp after trimming were discarded.
Reads were aligned and quantified using STAR (2.6.0a) to the Ensembl human transcriptome (hg38.p12, release 96).
Lowly abundant genes were removed as those <1 mean raw count or <1 mean count per million (CPM) across the dataset.
Differential gene expression was calculated using the voom function in limma.
Genome_build: GRCh38
Supplementary_files_format_and_content: Compiled_counts.txt: Tab-delimited text file containing STAR gene count values.

molecule: polyA RNA

growth_protocol: Cells were hormone-deprived via maintenance in medium supplemented with charcoal-stripped serum for 3 days prior to further treatment.

extract_protocol:
RNA extractions were performed using the RNeasy Mini kit (Qiagen).
Illumina Stranded mRNA Prep, for sequencing on NovaSEQ6000 with 2x150 paired-end reads.

treatment_protocol: After 3d hormone-deprivation, cells were transfected with the indicated siRNA (siNT = non-targeting control). 24hrs post-siRNA transfection, cells were treated with vehicle (0.01% EtOH) or 100pM estradiol. 24hrs after vehicle or E2 treatment, cells were collected for RNA extraction, processing, and sequencing.

tag: sirna

value: NT


title: KDM1A_WT_repl2

value: KDM1A (Abcam, 17721)


title: H3K27ac_SAMD1KO

value: H3K27ac (Active Motif, 39133)


title: TdgWT-Th2-1st-BS-rep2

data_processing:
The BS and PB reads were mapped to mouse genome mm9 using the BSMAP mapping tool. The mapping was done using the paired end datasets with the following parameter values: -p 4 -w 2 -v 5 -q 30 for BS-seq, and -p 4 -w 2 -v 0 -q 30 for PB-seq.
For each of the samples, the number of reads in which each cytosine within the amplicons was converted into thymine was counted.
For BS-seq, these counts were used to calculate the proportions for each cytosine to be nonmethylated/formylmethylated/carboxylmethylated (T), or methylated/hydroxymethylated (C).
For PB-seq, to compensate redundant reads derived from the opposite strand, number of reads standing for nonmethylated/methylated/hydroxymethylated (C) was multiplied by 0.5, and the proportions for each cytosine to be formylmethylated/carboxylmethylated (T) was calculated.
Genome_build: mm9
Supplementary_files_format_and_content: methylation_ratio .txt file


title: TdgKO-Th2-1st-BS-rep2

value: Tdg-iKO


title: TdgKO-Th2-1st-PB-rep1


title: H3K27ac_WT


title: DNMT3A1-WT_ChIP-seq_parental-rep-2

value: C3H embryo-derived mesenchymal progenitor cells

value: Parental line expressing FLAG-HA tagged DNMT3A1


title: MM330_siNT_E2_2


title: H3K4me3_SAMD1KO_repl1

value: H3K4me3 (Diagenode, C15410003)


title: BMDM-24h-BS-rep1

source_name: BMDMs

growth_protocol: 10 million bone marrow cells were seeded per 10 cm non-tissue culture plates in DMEM high glucose (50%) with 20% FBS, 30% L929-cell conditioned laboratory-made media (as source of M-CSF), and 100 U/ml penicillin/streptomycin+L-glutamine (GIBCO). After 4 days of differentiation, 20 ng/ml mouse M-CSF (Shenandoah Biotechnology) was added to the media. After an additional 2 days of culture, non-adherent cells were washed off with room temperature DMEM and macrophages were obtained as a homogeneous population of adherent cells which were scraped and subsequently seeded onto tissue culture-treated Petri dishes overnight in DMEM containing 20% FBS, 30% L929-cell conditioned laboratory-made media, and 100 U/ml penicillin/streptomycin+L-glutamine.

value: bone marrow derived macrophages


title: MM330_siNT_E2_1


title: MM330_siNT_3

description:
si_treatment: siNT
Compiled_counts.txt

value: Untreated


title: HFD_CANA_rep1

value: canagliflozin (30mg/kg)


title: BMDM-6h-BS-rep1


title: H3K4me3_WT_repl1

value: WT


title: DNMT3A1-delPWWP_ChIP-seq_sgDnmt3a

value: sgDnmt3a line expressing FLAG-HA tagged DNMT3A1 delPWWP


title: KDM1A_SAMD1KO_repl2


title: DNMT3A1-delPWWP_ChIP-seq_sgDnmt3a_input


title: H3K4me2_SAMD1KO_repl1

value: H3K4me2 (Diagenode, C15410035)


title: MM330_siNT_2

description:
si_treatment: siNT
Compiled_counts.txt


title: DNMT3A1-WT_ChIP-seq_parental-rep-2_input


title: DNMT3A1-D333N_ChIP-seq_sgEzh2_input

value: sgEzh2 line expressing FLAG-HA tagged DNMT3A1 D333N


title: MM330_siNT_1

description:
si_treatment: siNT
Compiled_counts.txt


title: H3K4me2_WT_repl1


title: DNMT3A1-D333N_ChIP-seq_sgEzh2


title: TetTKO-Th2-2nd-BS-rep1

source_name: Th2-2nd cells

growth_protocol: For the 2nd cycle of the cultivation, Th2-1st cells differentiated for 6 days were washed and cultured under resting conditions (medium without any cytokine) for 1 day. Cells were harvested, counted, and plated at 1M cells/ml for 2nd stimulation with plate-bound anti-CD3 (clone 2C11) and anti-CD28 (clone 37.51) antibodies at 1 μg/ml in the presence of 1000 U/ ml IL-4, 1 μg/ml anti-IFN-γ, for 1 day. On day 2 of the 2nd cycle, cells were removed from TCR/CD28 costimulation conditions and expanded in the presence of 20 U/mL IL-2 for 4 days.

value: TET-iTKO

value: T-cells from the spleen and lymphnodes


title: MM330_siMDC1_E2_3

description:
si_treatment: siMDC1_E2
Compiled_counts.txt


title: SAMD1_SAMD1KO_repl2

value: SAMD1 (self-made)


title: DNMT3A1-W330R_ChIP-seq_sgEzh2_input

value: sgEzh2 line expressing FLAG-HA tagged DNMT3A1 W330R


title: MM330_siMDC1_E2_2


title: TetTWT-Th2-1st-BS-rep1

value: wild type


title: DNMT3A1-W330R_ChIP-seq_sgEzh2

value: HA (ThermoFisher, #88836)


title: MM330_siMDC1_E2_1


title: Q001S0002B0016R3N

city: Ames

first: Roger

last: Wise

state: IA

address: Department of Plant Pathology, Entomology, and Microbiology, Iowa State University, 1344 Advanced Teaching & Research Bldg., 2213 Pammel Dr.

department: CICGRU

institute: USDA/ARS

description:
processed data file: Q001S0002B0016_salmon_counts.matrix_norm.csv
Sample 58
58_S58

data_processing:
library strategy: Amplicon-seq
Adapter Trimming - Each sample was processed using Trimmomatic to trim off adapters. Fastqc analysis revealed that most of the reads are of high quality. Hence, no quality trimming was performed.
Alignment - Reads from each sample was aligned to the genome of barley, Blumeria and vector sequences using STAR (dobin et. Al 2013).
Fusion read recognition - Custom python scripts was used to recognize fusion reads based on the partial alignment to the genomes and vector sequences. Vector portions were trimmed off from fusion reads.
Differential expression analysis - Salmon (Patro 2017) was used to obtain gene counts. Samples were size normalized and differential expression analysis was carried out using DESeq2 (Love 2014)
In-frame fusion reads  - Fusion reads expressed in-frame with the protein was recognized by using translating the entire sequence and comparing with the protein
Genome_build: Barley (Mascher 2017) and Blumeria (Ensembl Fungi Assembly EF 2) (Spanu 2018).
Supplementary_files_format_and_content: Read count data for barley and powdery mildew

organism:
Hordeum vulgare
Blumeria hordei

source_name: Barley infected first leaf (PO:0007094)

growth_protocol: Yeast were grown at 28C shaking at 225rpm. Cells were subcultured once they reached saturation (OD600 >2.5).

extract_protocol:
Yeast cells were treated with lyticase to digest cell walls and Y2H plasmids were extracted using alkaline lysis and precipitation.
Libraries were constructed using the Nugen Ultralow System V2 with Custom AnyDeplete to reduce the abundance of an amplicon common to all libraries using the manufacturer's instructions.

treatment_protocol: Yeast were incubated in SC-Leu-Trp (no selection) or SC-Leu-Trp-His (reporter selection) liquid media. Two rounds of growth selection were performed.

tag: plant genotype

value: CI 16151 (Resistant progenitor), m19089 (resistant bln1 mutant), m18982 (susceptible mla6 mutant), m19028 (susceptible mla6 + bln1 double mutant), m11526 (susceptible rar3 mutant)

tag: fungal isolate

value: Bgh isolate 5874 (AVRa6)

value: Non-selected

tag: tissue

value: Barley infected first leaf (PO:0007094)


title: DNMT3A1-R318W_ChIP-seq_sgEzh2

extract_protocol:
Cross-linking ChIP in MSCs was performed as described previously (Weinberg et al. 2019) using ~2x107 cells per immunoprecipitation. Prior to fixation, media was aspirated and cells washed once with PBS. Cells were cross-linked directly on the plate using 1% paraformaldehyde for 5 min at room temperature with gentle shaking. Glycine was added to quench (final concentration 125 mM, incubated for 5 min at room temperature), then cells were washed once with cold PBS, scraped off the plates, and pelleted. To obtain a soluble chromatin extract, cells were resuspended in 1 mL LB1 (50 mM HEPES, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100, 1x Complete protease inhibitor) and incubated rotating at 4°C for 10 min. Samples were centrifuged, resuspended in 1 mL LB2 (10 mM Tris-HCl pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1x Compete protease inhibitor), and incubated rotating at 4°C for 10 min. Finally, samples were centrifuged, resuspended in 1 mL LB3 (10 mM Tris-HCl pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% Na deoxycholate, 0.5% N-lauroylsarcosine, 1% Triton X-100, 1x Complete protease inhibitor), and homogenized by passing two times through a 27-gauge needle. Chromatin extracts were sonicated for 8 min (anti-HA ChIP) or 12 min (anti-histone PTM ChIP) using a Covaris E220 focused ultra-sonicator at peak power 140, duty factor 5, and cycles/burst 200. The lysates were incubated with 100 μl Pierce anti-HA beads (Themo Scientific, 88836) or 75 μl protein A Dynabeads (Invitrogen) bound to anti-H2AK119Ub (Cell Signaling, 8240) or anti-H3K27me3 (Cell Signaling Tech, 9733) antibodies and incubated overnight at 4°C with 5% kept as input DNA. Magnetic beads were sequentially washed with low salt buffer (150 mM NaCl; 0.1% SDS; 1% Triton X-100; 1 mM EDTA; 50 mM Tris-HCl), high salt buffer (500 mM NaCl; 0.1% SDS; 1% Triton X-100; 1 mM EDTA; 50 mM Tris-HCl), LiCl buffer (150 mM LiCl; 0.5% Na deoxycholate; 0.1% SDS; 1% Nonidet P-40; 1 mM EDTA; 50 mM Tris-HCl) and TE buffer (1 mM EDTA; 10 mM Tris-HCl). Beads were resuspended in elution buffer (1% SDS, 50 mM Tris-HCl pH 8.0, 10mM EDTA, 200 mM NaCl) and incubated for 30 min at 65°C. After centrifugation the eluate was reverse cross-linked overnight at 65°C. The eluate was then treated with RNaseA for 1 hr at 37°C and with Proteinase K (Roche) for 1 hr at 55°C and DNA was recovered using Qiagen PCR purification kit.
KAPA Hyper Prep kit

tag: cell type

value: C3H embryo-derived mesenchymal progenitor cells

tag: genotype

value: sgEzh2 line expressing FLAG-HA tagged DNMT3A1 R318W


title: DNMT3A1-R318W_ChIP-seq_sgEzh2_input


title: BMDM-0h-BS-rep1

tag: treatment

value: bone marrow derived macrophages


title: TdgKO-Th2-1st-BS-rep1

value: Tdg-iKO


title: TdgWT-Th2-1st-BS-rep1


title: Q001S0002B0016R2N

description:
processed data file: Q001S0002B0016_salmon_counts.matrix_norm.csv
Sample 46
46_S46


title: MM330_siFOXA1_E2_2

description:
si_treatment: siFOXA1_E2
Compiled_counts.txt


title: DNMT3A1-K299I_ChIP-seq_sgEzh2_input

value: sgEzh2 line expressing FLAG-HA tagged DNMT3A1 K299I


title: Q001S0002B0016R1N

description:
processed data file: Q001S0002B0016_salmon_counts.matrix_norm.csv
Sample 35
35_S35


title: Q001S0002B0016R3S

description:
processed data file: Q001S0002B0016_salmon_counts.matrix_norm.csv
Sample 64
64_S64

value: Selected


title: DNMT3A1-K299I_ChIP-seq_sgEzh2


title: TdgKO-Th2-2nd-BS-rep1


title: TetTWT-Th2-2nd-BS-rep1


title: TdgWT-Th2-2nd-BS-rep1


title: DNMT3A1-delPWWP_ChIP-seq_sgEzh2_input

value: sgEzh2 line expressing FLAG-HA tagged DNMT3A1 delPWWP


title: MM330_siER_E2_3

description:
si_treatment: siER_E2
Compiled_counts.txt

value: ER


title: Th2-BS-rep3

source_name: Th2 cells

value: T-cells from the spleen and lymphnodes


title: MM330_siER_E2_2


title: Q001S0002B0016R2S

description:
processed data file: Q001S0002B0016_salmon_counts.matrix_norm.csv
Sample 52
52_S52


title: TetTKO-Th2-1st-BS-rep1

value: TET-iTKO


title: Q001S0002B0016R1S

description:
processed data file: Q001S0002B0016_salmon_counts.matrix_norm.csv
Sample 40
40_S40


title: DNMT3A1-delPWWP_ChIP-seq_sgEzh2


title: Th2-BS-rep2


title: MM330_siER_E2_1


title: MM330_siFOXA1_E2_3


title: DNMT3A1-WT_ChIP-seq_sgEzh2_input

value: sgEzh2 line expressing FLAG-HA tagged DNMT3A1


title: Th2-BS-rep1

value: wild type


title: MM134_siNT_2

description:
si_treatment: siNT
Compiled_counts.txt

value: Untreated

value: NT


title: Naïve-BS-rep3

source_name: Naïve CD4 T-cells

growth_protocol: Total CD4+ T cells were isolated with the EasySep Mouse CD4+ T cell isolation kit (STEMCELL Technologies, Canada) from the spleen and lymph nodes. Then, CD4+CD25−CD62LhiCD44lo naive CD4+ T cells were FACS sorted.


title: Q001S0002B0015R1N

description:
processed data file: Q001S0002B0015_salmon_counts.matrix_norm.csv
Sample 34
34_S34

value: CI 16151 (Resistant progenitor), m19089 (resistant bln1 mutant), m18982 (susceptible mla6 mutant), m19028 (susceptible mla6 + bln1 double mutant), m11526 (susceptible rar3 mutant)

value: Bgh isolate 5874 (AVRa6)


title: MM330_siFOXA1_E2_1


title: MM134_siNT_3

description:
si_treatment: siNT
Compiled_counts.txt


title: Q001S0002B0015R2N

description:
processed data file: Q001S0002B0015_salmon_counts.matrix_norm.csv
Sample 45
45_S45

value: Non-selected

value: Barley infected first leaf (PO:0007094)


title: MM134_siNT_1

description:
si_treatment: siNT
Compiled_counts.txt


title: Q001S0002B0015R3S

description:
processed data file: Q001S0002B0015_salmon_counts.matrix_norm.csv
Sample 63
63_S63


title: DNMT3A1-WT_ChIP-seq_sgEzh2

value: HA (ThermoFisher, #88836)

value: C3H embryo-derived mesenchymal progenitor cells


title: Naïve-BS-rep2


title: DNMT3B-delPWWP-DNMT3A1-IDR_fusion_ChIP-seq_parental_input

value: Parental line expressing FLAG-HA tagged DNMT3B delPWWP fused with DNMT3A1 N-terminal IDR


title: MM134_siNT_E2_3


title: DNMT3B-delPWWP_ChIP-seq_parental_input

value: Parental line expressing FLAG-HA tagged DNMT3B delPWWP


title: MM134_siNT_E2_2


title: Q001S0002B0015R1S

description:
processed data file: Q001S0002B0015_salmon_counts.matrix_norm.csv
Sample 39
39_S39

value: Selected


title: Q001S0002B0015R2S

description:
processed data file: Q001S0002B0015_salmon_counts.matrix_norm.csv
Sample 51
51_S51


title: Q001S0002B0015R3N

description:
processed data file: Q001S0002B0015_salmon_counts.matrix_norm.csv
Sample 57
57_S57


title: Q001S0002B0012R2N

description:
processed data file: Q001S0002B0012_salmon_counts.matrix_norm.csv
Sample 56
56_S56


title: MM134_siNT_E2_1

description:
si_treatment: siNT_E2
Compiled_counts.txt


title: DNMT3A2-delPWWP_ChIP-seq_parental_input

value: Parental line expressing FLAG-HA tagged DNMT3A2 delPWWP


title: DNMT3B-delPWWP-DNMT3A1-IDR_fusion_ChIP-seq_parental