title: HeLa_rep1

description: replicates were merged for processing


title: ME24, replicate 3, scRNAseq

status: Public on May 11 2023

type: SRA

city: kunming

first: Ci

middle: bai

last: quan

address: bodalu

country: China

institute: lpbr

description: 10x Genomics

library_source: transcriptomic single cell

data_processing:
The demultiplexing, barcoded processing, gene counting and aggregation were made using the Cell Ranger software v4.0.0 (https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/what-is-cell-ranger)
Subsequently, unique molecular identifiers (UMI) matrices from each sample were merged in R and imported to Seurat(v4.1.1).
Assembly: Mmul_10/rheMac10
Supplementary files format and content: Tab-separated values files and matrix files

molecule: total RNA

organism: Macaca fascicularis

source_name: in vitro

extract_protocol:
Freshly collected DC and Si8 from RedMUC298trTg mice were flushed with ice-cold HBSSwo (Mg2+/Ca2+ free supplemented with 10mM HEPES, pH 7.2) to remove luminal content. The tissue was inverted and inflated by injection of HBSSwo. Epithelial isolation was performed by incubation of tissue with pre-digestion buffer (HBSSwo, 5 mM EDTA, 5% v/v FCS, 1 mM DTT) in a shaking incubator. The isolated epithelial cells were resuspended in digestion buffer (HBSS with Mg2+/Ca2+ supplemented with 2 mg/ml collagenase type I and 40 U/ml DNase I) and incubated at 37 °C for 30 min. Cells were washed and re-suspended in PBS and stained with a Viability Dye, washed and resuspended in FACS buffer (HBSSwo, 2% v/v FCS, 5mM EDTA). Alive GCs were sorted using a FACS Jazz (Becton Dickinson) according to the presence or absence of mCherry signal using a C57Bl6 mouse as negative control. Goblet cells were washed in HBSS-/- and resuspended in HBSS-/- supplemented with 0.04% w/v BSA.
Library was performed according to the manufacter’s instructions (Chromium Single Cell 3’ Reagent Kit v3.1 protocol, 10x Genomics). Briefly, GCs were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip G to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was retrotranscripted to cDNA, which contains an Ilumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the apropiated sized fragments were selected with SPRIselect reagent for subsequent library construction. On average, approximately 13,200 single cells were loaded on each channel and approximately 8,000 cells were recovered. Libraries were sequenced on the Illumina NovaSeq 6000 platform

tag: cell type

value: Embryo

tag: tissue

value: in vitro

tag: age


title: 171: P17, colon, tumor, anti-PD-1, scRNA-seq

city: Guangzhou

first: Cheng

last: Wu

address: No. 74, Zhongshan 2nd Road, Yuexiu District, Guangzhou

institute: Sun Yat-sen University

description: 10x Genomics

data_processing:
The demultiplexing, barcoded processing, gene counting and aggregation were made using the Cell Ranger software vcellranger-5.0.1 (https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/what-is-cell-ranger)
Assembly: CRCh38
Supplementary files format and content: Tab-separated values files and matrix files

organism: Homo sapiens

source_name: colon

extract_protocol:
Tumor biopsy samples were first stored in liquid nitrogen with the SenotechTM Tissue Storage Buffer(#JZ-SC-5802，Senotech Genomics, Shanghai, China)or proceed directly to tissue disassociation. Heat the frozen tissue in a 37 ° C water bath,then transfer the tissue samples together with the freezed-storage solution to a new 15mL centrifuge tube, add 5mL preheated RPMI-1640 medium (Gibco).Centrifuge at room temperature at 300×g for 5min to remove the supernatant without disturbing the tissue sample at the bottom of the centrifuge tube.The tissue samples enzymatically digested with Neuronal Isolation Enzyme(Thermo Scientific) for 20-30 min at 37 C according to the manufacturer’s protocol.The dissociated cells were next passed through a 40-mm cell-strainer (BD) in the RPMI-1640 medium (Invitrogen) with 10% FBS until uniform cell suspensions were obtained. Subsequently, the suspended cells were passed through cell strainers and centrifuged at 400 g for 10 min. Red blood cells were removed via the same procedure described above. The recommended cell washing and resuspension solution is 1X PBS (calcium and magnesium free) containing 0.04% weight/volume BSA (400 µg/ml).
Single-cell RNA-seq libraries were prepared using the Chromium Next GEM Single Cell 3’ Kit v2 or v3 from 10x Genomics, following the manufacturer’s instructions. In brief, single cells were  resuspended in PBS containing 0.04% BSA to a final concentration of 500~1,200 cells/ml as determined by Countess 3 Automated Cell Counters(Invitrogen). ~10,000 cells were captured in droplets to generate nanoliter-scale Gel bead in EMulsion (GEMs). GEMs were then reverse transcribed in a C1000 Touch Thermal Cycler (Bio-Rad) programmed at 53C for 45 min, 85C for 5 min, and held at 4C. After reverse transcription and cell barcoding, emulsions were broken and cDNA was isolated and purified with Cleanup Mix containing DynaBeads and SPRIselect reagent (Thermo Fisher Scientific), followed by PCR amplification. Amplified cDNA was fragmented and end-repaired, double-sided size-selected, PCR-amplified with sample indexing primers, and double-sided size-selected or RNA-seq library construction. Libraries prepared according to the manufacturer’s user guide were then purified and profiled for quality assessment. Single-cell RNA libraries were sequenced by an Illumina NovaSeq 6000 or BGISEQ DNBSEQ-T7 sequencer with 150 bp paired-end reads.

tag: subject

value: Goblet cells

value: colon

tag: genotype

value: tumor

tag: treatment


title: ME25, replicate 3, scRNAseq

description: 10x Genomics


title: ME23, replicate 4, scRNAseq

description: 10x Genomics


title: B16_dLN_aCD4_UT_CD8_5

city: Noda

first: Hiroyasu

last: Aoki

state: Chiba

address: 2669 Yamazaki

department: Research Institute for Biomedical Sciences

country: Japan

institute: Tokyo University of Science

description:
The Fscn1 knockin (Fscn1-KI) mouse (Accession No. CDB0154E:  http://www2.clst.riken.jp/arg/mutant%20mice%20list.html) was generated by CRISPR/Cas9-mediated knockin in zygotes as previously described (Abe et al., 2020). For the homologous recombination-mediated knockin, the donor vector consisting of homology arms and P2A-DTR-P2A-Cre (insert gene) was generated to insert the P2A-DTR-P2A-Cre cassette at the 3-base upstream of the PAM sequence. For microinjection, the mixture of crRNA (CRISPR RNA) (50ng/ul), tracrRNA (trans-activating crRNA) (100ng/ul), donor vector (10ng/ul) and Cas9 protein (100ng/ul) were injected into the pronucleus of one-cell stage zygote. From 233 zygotes, 57 F0 mice were obtained and 3 of them were knockin mice confirmed by PCR, in which the PCR fragments of KI allele with 5’FW and 5’REV primers (1301 bp), and 3’FW and 3’REV primers (587 bp) were detected. The germline transmissions of knockin F0 mice were confirmed by genotyping of F1 mice. gRNA target sequences and primers used for genotyping are shown in Supplementary Table 1.
Recipient B6 mice were lethally irradiated (8.5 Gy, split into 2 doses given 3 hours apart) the day before transplantation with 3 × 106 Fscn1-KI BM cells. Mice were used for the experiments at least 8 weeks after transplantation.

library_source: transcriptomic

data_processing:
Data processing of Bulk TCRseq was performed as described in Aoki et al. (Aoki et al., 2021).
Adapter trimming and quality filtering of sequencing data were performed by using Cutadapt-3.2  (Martin et al., 2011) and PRINSEQ-0.20.4 (Schmieder et al., 2011). Sequencing data were processed by MiXCR-3.0.5 (Bolotin et al., 2015). In MiXCR, filtered reads were aligned to reference mouse TCR V/D/J sequences registered in the international ImMunoGeneTics (IMGT) information system: -vParameters.geneFeatureToAlign = VTranscript -vjAlignmentOrder = JThenV, then identical sequences were assembled and grouped in clones with PCR and sequencing error correlation with the following parameters: -badQualityThreshold=10, –separateByV=true, --only-productive=true, –region-of-interest=CDR3. The Variable (V) and Joining (J) segment of TCRs were represented in IMGT gene nomenclature.
List of final clones were analyzed by VDJtools-1.2.1 (Shugay et al., 2015). Then, the sequencing reads of sample was normalized to the cell count in each sample by “DownSample” command of VDJtools. T-cell clones were determined as TCR reads with the same TCR V segment, J segment and CDR3 nucleotide sequence.
Assembly: IMGT reference sequences of mouse TRA and TRB
Supplementary files format and content: Text files in VDJtools format include read count and frequency, complementarity determining region 3 (CDR3) nucleotide and amino acid sequences, Variable(V)/ Diversity(D)/ Joining(J) segment names, and V, D and J segment boundaries within CDR3 nucleotide sequence of individual clones

organism: Mus musculus

source_name: dLN on day 14 after innoculation of B16

extract_protocol:
Intravascular leukocytes were stained by i.v. injection of FITC-conjugated anti-CD45.2 mAb (3 mg/mouse, clone 104, BioLegend) three minutes before sacrifice.
dLN was digested for 45 minutes at 37°C with 0.1% collagenase (FUJIFILM Wako) then subjected to 25% Percoll PLUS (Cytiva) gradient and leukocytes were recovered from the pellet.
Tumors were cut into small fragments and digested for 45 minutes at 37°C with 0.1% collagenase (FUJIFILM Wako), then subjected to 40% Percoll PLUS (Cytiva) and Histopaque-1083 (Sigma-Aldrich) gradient, and leukocytes were recovered from the interphase. The cell concentration of the suspensions was determined using Flow-Count fluorospheres (Beckman Coulter) and a Cytoflex flow cytometer (Beckman Coulter).
Cells were stained with a mix of Fc Block (anti-mouse CD16/CD32 mAb; clone 93, BioLegend) and fluorophore-conjugated anti-mouse mAbs. For dLN samples, Lineage+ (CD11b, B220, NK1.1, Ter119) cells were depleted using BD IMag Streptavidin Particles Plus-DM (BD Biosciences). For tumor samples, TCRb+ T cells were enriched magnetically using Anti-APC MicroBeads (Miltenyi) and LS columns (Miltenyi).
CD8+ T cells from tumor, and CD8+ CD44high T cells from dLN were sorted using FACS Aria II or Aria III (BD Biosciences). Nonviable cells were excluded from the analysis based on forward and side scatter profiles and propidium iodide staining. Intravascular leukocytes were also excluded. Purity of sorted cells was always over 95%.
Bulk TCR sequencing library was prepared according to the previous report (Tsunoda et al., 2021). In brief, to perform reverse transcription and template-switching, mRNA-trapped oligo-dT-immobilized Dynabeads M270-streptavidin (Thermo Fisher Scientific) were suspended in 10 µL of RT mix [1× First Strand buffer (Thermo Fisher Scientific), 1 mM dNTP, 2.5 mM DTT (Thermo Fisher Scientific), 1 M betaine (Sigma-Aldrich), 9 mM MgCl2 (NIPPON GENE), 1 U/µL RNaseIn Plus RNase Inhibitor (Promega), 10 U/µL Superscript II (Thermo Fisher Scientific), and 1 µM of i5-TSO], and incubated for 60 min at 42ºC and immediately cooled on ice. To amplify the TCR cDNA containing complementarity determining region 3 (CDR3), nested PCR of the TCR locus was performed following the purification of PCR product by an Agencort AM Pure XP kit (Beckman Coulter) at a 0.7:1 ratio of beads to sample and eluted with 20 µL of 10 mM Tris-HCl (pH 8.0). To amplify TCR libraries and add adaptor sequences for the next-generation sequencer, the third PCR was performed. The third-PCR products were purified as second PCR. The products were pooled and then purified and subjected to dual size selection using ProNex size-selective purification system (Promega) and eluted with 25 μL of 10 mM Tris-HCl (pH 8.5). Final TCR libraries, whose lengths were about 600 base pairs were sequenced using an Illumina Novaseq 6000 S4 flowcell (67 bp read 1 and 140 bp read 2) (Illumina). Only read2 contained the sequence regarding the definition of T cell clones.
Bulk TCR-seq

treatment_protocol: B16F10 cells (5 × 105 cells) were inoculated subcutaneously (s.c.) into the right flanks of the Fscn1-KI BMC mice. Anti-PD-L1 mAb (clone 10F.9G2, BioLegend) or Anti-CD4 mAb (clone GK1.5, BioLegend) was injected intraperitoneally (i.p.) at a dose of 200 μg per mouse on days 5 and 9 after tumor inoculation.

tag: strain

value: Bone marrow chimera of Fscn1 KI

value: anti-CD4 mAbs

value: Intravenous (i.v.) staining-negative, lineage-negative (CD11b, B220, NK1.1 and Ter119), TCRbeta+, CD4- CD8+ and CD44high T cells

value: 10 weeks post bone marrow transplantation

tag: Sex

value: female

tag: tumour cell line

value: B16F10 melanoma

tag: organ

tag: date


title: B16_dLN_aPDL1_DT_CD8_3

value: anti-PD-L1 mAbs with DT


title: ME23, replicate 3, scRNAseq

description: 10x Genomics


title: B16_dLN_aPDL1_DT_CD8_5


title: IFNg Treated Replicate 4

city: Munich

first: Moritz

middle: J

last: Rossner

address: Nussbaumstr. 7

department: Psychiatry

country: Germany

data_processing:
FastQC for quality check
TMAP Aligner was used to map reads against Mus musculus (mm10) and ENSEMBL annotation 86 was used to quantify genes on a partek flow software
DESeq2 R package was used to perform differential expression
Assembly: Mus Musculus (mm10)
Supplementary files format and content: Tab delimited Count file

source_name: Spinal Cord

growth_protocol: For all assays, microglia were obtained from 2-3 month old male C57BL6 mice induced with EAE.

extract_protocol:
RNAeasy Micro Kit (Qiagen) was used to extract RNA from sorted cells
1 ul of ERCC RNA Spike-In Mix (ThermoFisher, Carlsbad, California, US) diluted 1:5000 was added to the isolated RNA as an external control. cDNA was synthesized using Ovation RNA-Seq System V2 (NuGen, Groningen, Netherlands). 100 ng of cDNA was used as input for fragmentation and followed by library preparation using the IonXpress plus gDNA and Amplicon Library preparation kit (ThermoFisher, Carlsbad, California, US) as described by the manufacturer. The library was then size selected on a 2% E-Gel (ThermoFisher, Carlsbad, California, US). Sample specific barcodes were then added and amplified. Individual sample libraries were quantified using a Kapa Library Quantification Kit (Kapa, Wilmington, Massachusetts, US) using samples diluted 1:200. Equal quantities of individual samples were then pooled and sequenced on an Ion Proton Sequencer.

treatment_protocol: At day 15 after EAE induction mice were treated with IFN-g or its vehicle (PBS) for five days during the effector phase of disease.

tag: disease

value: experimental autoimmune encephalomyelitis (EAE)

value: Spinal Cord

value: male

value: Microglia

value: IFNg


title: B16_dLN_aCD4_UT_CD8_4


title: B16_dLN_aCD4_UT_CD8_3


title: ME23, replicate 2, scRNAseq

description: 10x Genomics

value: Embryo


title: PBS Treated Replicate 4

value: PBS


title: IFNg Treated Replicate 3


title: K562_rep2

city: Beijing

first: Yuanchao

last: Xue

address: Datun Road #15

department: Institute of Biophysics

institute: Chinese Academy of Sciences

description:
replicates were merged for processing
EP_and_PP.K562.network.xls

data_processing:
Library strategy: RIC-seq
Illumina bcl2fastq (v2.16) conversion software used for basecalling.
After sequencing, RIC-seq reads were aligned to the human genome using the RICpipe program with minor revisions. Briefly, RIC-seq reads were first mapped to the reference genome (assembly version: hg19 for human and mm9 for mouse) using the STAR software (v2.5.2b). The parameters were set as: --runMode alignReads --genomeDir index --readFilesIn read.fq --outFileNamePrefix prefix --outReadsUnmapped Fastx --outFilterMultimapNmax 100 --outSAMattributes All --alignIntronMin 1 --scoreGapNoncan -4 --scoreGapATAC -4 --chimSegmentMin 15 --chimJunctionOverhangMin 15 --limitOutSJcollapsed 10000000 --limitIObufferSize 1500000000 --runThreadN 16 --alignSJoverhangMin 15 --alignSJDBoverhangMin 10 --alignSJstitchMismatchNmax 5 -1 5 5 --outFilterMatchNminOverLread 0.5 --outFilterScoreMinOverLread 0.5. Next, unmapped reads reported by STAR were aligned again to the reference genome by the bwa program (v0.7.17) with the following parameters: bwa mem -k 12 -T 15. Alignments from STAR and bwa were combined and sent to RICpipe for the subsequent analysis to extract chimeric reads.
Enhancer and promoter intervals for each cell are defined as previously described (Cai et al. Nature, 2020, 582(7812): 432-437.), using ChIP-seq data of H3K4me3, H3K4me1 and H3K27ac downloaded from the ENCODE or GEO database.
Using these defined promoter and enhancer regions, we adopted a Monte Carlo simulation strategy to detect significant enhancer-promoter interactions (P < 0.05, unique chimeric reads ≥ 2) as previously described.
Genome_build: hg19, mm9
Supplementary_files_format_and_content: enhancer-promoter and promoter-promoter interaction networks in Excel format

source_name: human K562 cells

growth_protocol: GM12878 and K562 cells were grown in RPMI 1640 supplemented with 10% fetal bovine serum and 100 U/ml penicillin/streptomycin (Life Technologies; 15140). IMR-90 cells were grown in DMEM/F12 + Glutamax (Life Technologies; 10565) supplemented with 5 ng/ml fibroblast growth factor-basic (R&D), 1 μg/ml hydrocortisone hemisuccinate, 50 μg/ml ascorbic acid, 5 μg/ml rhInsulin, 2% fetal bovine serum and 100 U/ml penicillin/streptomycin. Human embryonic stem cells (WA01, Line H1) were obtained from Dr. Hongjie Yao laboratory and cultured according to the protocol from WiCell Research Institute. The hESCs were maintained in mTeSR1 medium (Stem Cell Technologies) in feeder-free conditions on hESC qualified Matrigel (BD Biosciences). mTeSR1 media was changed daily and cells were passaged every 6-8 days with 1 ml of ReLeSR (Stem Cell Technologies). hNPC cells were grown in DMEM/F12 + Glutamax (Life Technologies; 10565) supplemented with 20 ng/ml fibroblast growth factor-basic (R&D), 0.5× N-2, 0.5× B-27,  and 100 U/ml penicillin/streptomycin. MEF and HepG2 cells were grown in DMEM containing 10% fetal bovine serum plus 100 U/ml penicillin/streptomycin.

extract_protocol:
Cells were treated with micrococcal nuclease and proteinase K. Total RNA was extracted with TRIzol LS.
Total RNA was extracted, and rRNA was depleted with specific probes. After fragmentation, the biotin-marked RNA fragments were enriched with streptavidin magnetic beads, and converted into Illumina HiSeq X10 compatible libraries.

treatment_protocol: Cells were fixed with  PBS containing 1% formaldehyde, and then treated with 1/20 volume of 2.5 M glycine.

tag: cell line


title: B16_dLN_aPDL1_DT_CD8_2


title: B16_dLN_aCD4_UT_CD8_2


title: IMR90_rep1

description:
replicates were merged for processing
IMR90.Enhancer_Promoter.chimeric_fragment.bed

source_name: human IMR90 cells


title: hNPC_rep2

description:
replicates were merged for processing
EP_and_PP.hNPC.network.xls

source_name: human NPC cells

value: hNPC


title: hNPC_rep1


title: Not Immunized Mice Replicate 2

value: Not Immunized


title: ME23, replicate 1, scRNAseq

description: 10x Genomics

value: in vitro


title: PBS Treated Replicate 3


title: ME24, replicate 1, scRNAseq

description: 10x Genomics


title: HepG2_rep2

description:
replicates were merged for processing
EP_and_PP.HepG2.network.xls

source_name: human HepG2 cells


title: B16_dLN_aCD4_UT_CD8_1

value: Bone marrow chimera of Fscn1 KI


title: ME22, replicate 3, scRNAseq

description: 10x Genomics


title: K562_rep1


title: B16_dLN_aPDL1_DT_CD8_1


title: ME22, replicate 2, scRNAseq

description: 10x Genomics


title: Not Immunized Mice Replicate 1


title: IFNg Treated Replicate 2


title: IMR90_rep2


title: HepG2_rep1


title: H1_rep2

description:
replicates were merged for processing
H1.Enhancer_Promoter.chimeric_fragment.bed

source_name: human H1 cells


title: B16_dLN_aCD4_DT_CD8_3

value: anti-CD4 mAbs with DT


title: ME22, replicate 1, scRNAseq

description: 10x Genomics


title: H1_rep1


title: B16_dLN_aCD4_DT_CD8_2

value: Intravenous (i.v.) staining-negative, lineage-negative (CD11b, B220, NK1.1 and Ter119), TCRbeta+, CD4- CD8+ and CD44high T cells


title: PBS Treated Replicate 1


title: IFNg Treated Replicate 1


title: ME21, replicate 2, scRNAseq

description: 10x Genomics


title: GM12878_rep2

description:
replicates were merged for processing
EP_and_PP.GM12878.network.xls

source_name: human GM12878 cells


title: B16_dLN_aCD4_DT_CD8_1

value: 10 weeks post bone marrow transplantation


title: ME21, replicate 1, scRNAseq

description: 10x Genomics


title: B16_dLN_CD4PDL1_UT_CD8_4

value: combination of anti-CD4 and anti-PD-L1 mAbs

value: female


title: Rhino 1, Replicate 2

city: Escondido

first: Elena

last: Ruggeri

state: CA

address: 15600 San Paqual Valley Rd.

country: USA

institute: San Diego Zoo Wildlife Alliance

data_processing:
Galaxy Web Platform
Sequence reads were trimmed for adaptor sequence/low-quality sequence using Trimmomatic
Trimmed sequence reads were mapped to  CerSimCot1.0 using HISAT2
Read count extraction was performed with StringTie
Assembly: CerSimCot 1.0, Chromosome_Locations.xlsx contains the coordinates for all MSTRG IDs
Supplementary files format and content: tab-delimited text files include count values for each Sample

organism: Ceratotherium simum simum

source_name: Granulosa Cell

extract_protocol:
Total RNA was isoalted using the PicoPure RNA Isolation Kit (Thermo fisher Scientific)
RNA libraries were prepared with the NEBNext® Ultra™ II RNA Library Prep Kit for Illumina

value: Granulosa Cell

value: WT

value: in vivo

tag: time

value: Pre IVM


title: Rhino 2, Replicate 3


title: ME20, replicate 1, scRNAseq

description: 10x Genomics


title: ME20, replicate 2, scRNAseq

description: 10x Genomics


title: ME19, replicate 3, scRNAseq

description: 10x Genomics


title: GM12878_rep1


title: HC,rep8,scRNA_seq

city: Hangzhou

first: jianpeng

last: sheng

address: No. 79 Qingchun Street

institute: Zhejiang University School of Medicine

description: 10x Genomics

data_processing:
The demultiplexing, barcoded processing, gene counting and aggregation were made using the Cell Ranger software (https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/what-is-cell-ranger).
Assembly: GRCh38
Supplementary files format and content: Tab-separated values files and matrix files (barcodes.tsv, features.tsv, matrix.mtx).

source_name: Healthy control colon

extract_protocol:
Biopsy specimens were collected into RPMI 1640 medium on ice and processed immediately into single cells. Single cells were dissociated from the intestinal samples obtained by biopsies through the method mentioned in the FACS analysis.
The single cells were loaded into Chromium microfluidic chips with v3 chemistry, then barcoded with a 10× Chromium Controller (10X Genomics, Pleasanton, CA, USA). RNA from the barcoded cells was subsequently reverse-transcribed and sequencing libraries were constructed with reagents from a Chromium Single Cell v3 reagent kit (10X Genomics) according to the manufacturer’s instructions.

value: Healthy control colon

value: Healthy

tag: sample group

value: HC

tag: individual


title: B16_dLN_aCD4_DT_CD8_4

value: B16F10 melanoma


title: B16_dLN_CD4PDL1_UT_CD8_2


title: B16_dLN_CD4PDL1_UT_CD8_3


title: HC,rep7,scRNA_seq

description: 10x Genomics


title: Rhino 1, Replicate 3


title: PBS Treated Replicate 2

value: experimental autoimmune encephalomyelitis (EAE)

value: Spinal Cord

value: male

value: Microglia

value: PBS


title: B16_dLN_CD4PDL1_UT_CD8_1

value: Bone marrow chimera of Fscn1 KI

value: Intravenous (i.v.) staining-negative, lineage-negative (CD11b, B220, NK1.1 and Ter119), TCRbeta+, CD4- CD8+ and CD44high T cells


title: Rhino 2, Replicate 1


title: ME19, replicate 2, scRNAseq

description: 10x Genomics

value: Embryo

value: in vitro


title: HC,rep6,scRNA_seq

description: 10x Genomics


title: B16_dLN_CD4PDL1_DT_CD8_4

value: combination of anti-CD4 and anti-PD-L1 mAbs with DT

value: 10 weeks post bone marrow transplantation

value: female


title: HC,rep5,scRNA_seq

description: 10x Genomics


title: Rhino 1, Replicate 1

value: Granulosa Cell

value: WT

value: in vivo

value: Pre IVM


title: B16_dLN_CD4PDL1_DT_CD8_3

value: B16F10 melanoma


title: Rhino 2, Replicate 2


title: UC,rep4,scRNA_seq

description: 10x Genomics

source_name: Inflamed colon region

value: Inflamed colon region

value: Ulcerative colitis

value: UC


title: bacteria+macrophage at t=285min; rep4

city: Basel

first: Lucia

last: Vidakovic

address: Spitalstrasse 41

country: Switzerland

institute: Biozentrum, University of Basel

description: sample has been re-sequenced

data_processing:
Sequencing of the cDNA libraries was performed by Vertis Biotechnologie AG.
Fastq reads were aligned to a reference genome Vibrio cholerae O1 biovar El Tor str. N16961 using STAR (version 2.5.3a).
Read counts were calculated using STAR  (version 2.5.3a).
Data were normalized to a library size factor, calculated by dividing the total number of reads from each sample to the median of the total number of reads across all samples
Data were transformed to a log2 scale
A minimal expression threshold was set to 5
Genome_build: Vibrio cholerae O1 biovar El Tor str. N16961
Genome_build: gtf file hg_v_comb.gtf
Supplementary_files_format_and_content: Expression data sets: csv files include reads counts before and after normalization. raw_count_data.csv - raw counts from each sample, final_normalized_data.csv - normalized, log2 transformed, with minimal value set to 5,gtf file hg_v_comb.gtf - gtf used.
Supplementary_files_format_and_content: V_cholerae_significantly_up_and_downregulated_genes.xlsx: Excel file includes differentially expressed genes in V. cholerae: numbers in each row indicate the log2 fold change calculated for each sample from an average of all naive samples (bacteria only at t = 0 min).

organism:
Vibrio cholerae O1 biovar El Tor
Homo sapiens

source_name: whole bacterial cells and macrophages

growth_protocol: Vibrio cholerae C6706 WT was exposed to THP-1 derived macrophages in a well of a 12-well plate for a defined time at 37°C and 5% CO2.

extract_protocol:
RNA was isolated using the mirVana Kit(Ambion, cat. no. AM1560) according to the manufacturer`s protocol. The rRNA was depleted using the Ribo-Zero rRNA Removal Kit from Illumina. The RNA integrity was checked on a Shimadzu MultiNA microchip electrophoresis system.
cDNA libraries were prepared by adapter ligation, cDNA synthesis using the M-MLV reverse transcriptase and PCR amplification of the resulting cDNA. Quality of the prepared libraries was tested using the Shimadzu MultiNA microchip electrophoresis system.

treatment_protocol: At specific time points after start of the co-incubation of V. cholerae and THP-1 derived macrophages RNAlater was added to the well. Afterwards the sample was scraped off the surface of the well and stored until RNA was isolated.

tag: bacterial strain

tag: genotype (bacteria)

value: Wild type

tag: human cell line

value: ATCC TIB-202

tag: human cell type

value: THP-1 derived macrophages


title: ME18, replicate 3, scRNAseq

description: 10x Genomics

tag: cell type

tag: tissue

tag: age


title: ME18, replicate 4, scRNAseq

description: 10x Genomics


title: B16_dLN_CD4PDL1_DT_CD8_2

tag: strain

tag: genotype

value: Bone marrow chimera of Fscn1 KI

tag: treatment

value: Intravenous (i.v.) staining-negative, lineage-negative (CD11b, B220, NK1.1 and Ter119), TCRbeta+, CD4- CD8+ and CD44high T cells


title: bacteria+macrophage at t=285min plankt.; rep4

description: sample has been re-sequenced


title: ME18, replicate 2, scRNAseq

description: 10x Genomics

value: Embryo

value: in vitro


title: ME18, replicate 1, scRNAseq

description: 10x Genomics


title: UC,rep2,scRNA_seq

description: 10x Genomics


title: UC,rep3,scRNA_seq

description: 10x Genomics


title: bacteria+macrophage at t=265min; rep4

description: sample has been re-sequenced


title: UC,rep1,scRNA_seq

description: 10x Genomics


title: B16_Tumor_aPDL1_UT_CD8_5

source_name: tumor on day 14 after innoculation of B16

value: anti-PD-L1 mAbs

value: Intravenous (i.v.) staining-negative, lineage-negative (CD11b, B220, NK1.1 and Ter119), TCRbeta+ and CD4- CD8+ T cells

value: 10 weeks post bone marrow transplantation

tag: Sex

tag: tumour cell line

tag: organ


title: B16_Tumor_aPDL1_UT_CD8_4

value: female

tag: date


title: High-fat diet fed WT mice, shYap, Tumor, rep3

city: Los Angeles

first: Ekihiro

last: Seki

address: 8700 Beverly Blvd

department: Medicine

institute: Cedars-Sinai Medical Center

description:
HFD-SH-4
Partek_HFMetYap_Quantify_to_annotation_model_(Partek_E_M)_Gene_counts.txt

data_processing:
instrument model: BGISEQ/DNBSEQ-G400
STAR(ver 2.7.3a) was used to align sequencing reads to the reference mouse genome (GRCm38/mm10) via Partek Flow software.
The relative gene expression read counts were mapped using Partek E/M to quantify to the annotation model, mm10. (Ensembl Transcropts release 99)
DESeq2 was used to conduct differential gene expression analysis using read counts and the median of ratios method.
GSEA was performed using GSEA software (Broad Institute)
Assembly: mm10
Supplementary files format and content: tab-delimited text files include raw count values for each sample

source_name: Liver Tumor

extract_protocol:
Total RNA was extracted from metastatic liver tumor tissues using TRIxol and purified using a NucleoSpin RNA kit in accordance with the manufacturer's instructions.
Library construction on the platform includes fragmentation, size selection, end repair and A-tailing, adaptor ligation, PCR amplification, and splint circularization.

treatment_protocol: Liver metastasis was induced by splenic injection of CRC with or without tumor, and tumor tissue was collected for RNAsequencing

value: Liver Tumor

tag: cell line

value: CRC liver metastasis

value: shYap

value: High-fat diet, shYap, liver metastasis


title: bacteria+macrophage at t=265min plankt.; rep4

description: sample has been re-sequenced


title: UC-self control,rep4,scRNA_seq

description: 10x Genomics

source_name: Normal ascending colon

value: Normal ascending colon

tag: disease

value: Ulcerative colitis

tag: sample group

value: UC-self control


title: bacteria+macrophage at t=235min; rep4

description: sample has been re-sequenced

tag: bacterial strain

value: Wild type

value: ATCC TIB-202


title: B16_Tumor_aPDL1_UT_CD8_3

value: Bone marrow chimera of Fscn1 KI

value: B16F10 melanoma


title: High-fat diet fed WT mice, shYap, Tumor, rep4

description:
HFD-SH-5
Partek_HFMetYap_Quantify_to_annotation_model_(Partek_E_M)_Gene_counts.txt


title: bacteria+macrophage at t=195min; rep4

description: sample has been re-sequenced

tag: genotype (bacteria)

value: THP-1 derived macrophages


title: UC-self control,rep3,scRNA_seq

description: 10x Genomics

tag: individual


title: High-fat diet fed WT mice, shYap, Tumor, rep2

description:
HFD-SH-2
Partek_HFMetYap_Quantify_to_annotation_model_(Partek_E_M)_Gene_counts.txt


title: ME19, replicate 1, scRNAseq

description: 10x Genomics

source_name: in vitro

value: Embryo

value: in vitro


title: B16_Tumor_aPDL1_UT_CD8_2

value: anti-PD-L1 mAbs

value: Intravenous (i.v.) staining-negative, lineage-negative (CD11b, B220, NK1.1 and Ter119), TCRbeta+ and CD4- CD8+ T cells


title: UC-self control,rep2,scRNA_seq

description: 10x Genomics


title: bacteria+macrophage at t=120min; rep4

description: sample has been re-sequenced

tag: human cell line

tag: human cell type


title: High-fat diet fed WT mice, shCon, Tumor, rep4

description:
HFD-NC-4
Partek_HFMetYap_Quantify_to_annotation_model_(Partek_E_M)_Gene_counts.txt

tag: tissue

tag: cell type

value: shControl

value: High-fat diet, shCon, liver metastasis


title: UC-self control,rep1,scRNA_seq

description: 10x Genomics


title: B16_Tumor_aPDL1_UT_CD8_1

tag: strain

tag: age

value: 10 weeks post bone marrow transplantation

value: female


title: High-fat diet fed WT mice, shYap, Tumor, rep1

description:
HFD-SH-1
Partek_HFMetYap_Quantify_to_annotation_model_(Partek_E_M)_Gene_counts.txt

value: Liver Tumor

value: CRC liver metastasis

tag: genotype

value: shYap

tag: treatment

value: High-fat diet, shYap, liver metastasis


title: B16_Tumor_aPDL1_DT_CD8_5

value: anti-PD-L1 mAbs with DT

tag: Sex

tag: tumour cell line


title: bacteria+macrophage at t=285min; rep3

description: sample has been re-sequenced

value: Wild type

value: ATCC TIB-202


title: B16_dLN_CD4PDL1_DT_CD8_1

value: Bone marrow chimera of Fscn1 KI

value: combination of anti-CD4 and anti-PD-L1 mAbs with DT

value: Intravenous (i.v.) staining-negative, lineage-negative (CD11b, B220, NK1.1 and Ter119), TCRbeta+, CD4- CD8+ and CD44high T cells

value: B16F10 melanoma

tag: organ

tag: date


title: hCD4 STARR Donor IV _2

city: Cincinnati

first: Kurtis

last: Stefan

state: OH

address: 3333 Burnet Ave

department: Dept Allergy & Immunology

institute: University of Cincinnati

data_processing:
Fastq are concatenated for each biologic donor before alignment.
Trimming the adapters with TrimGalore. This step is particularly important when the reads are long and the fragments are short-resulting in sequencing adapters at the end of read. If adapter is not removed the read will not map. TrimGalore can recognize standard adapters, such as Illumina or Nexterra/Tn5 adapters.
Mapping reads with BowTie. Only uniquely mapped reads with less than 3 mismatches are used in the downstream analysis. Results are saved as a .bam file. (Bam files are downloaded for further processing)
The per duplication count of each unique read for each biologic donor was fitted to the negative binomial distribution for each technical replicate (input plasmid library). Duplicate reads exceeding the 75th percentile were removed from each sample by MACS2 to reduce false discovery of enhancers resulting from PCR duplication. Enhancers were called using MACS2 in which the plasmid reads were used as a control, [macs2 callpeak -f BEDPE --keep-dup all -g 2.7E9], and with -log10(P-value) greater than 75. NRE were also detected with MACS2, using the plasmid reads as treatment and the STARR RNA as control [macs2 callpeak -f BEDPE --keep-dup all -g 2.7E9], with logP greater than 30. Both enhancers and silencers are retained only if intersecting an ATAC-Seq narrowPeak called using MACS2 from the plasmid reads alone.
Assembly: hg19
Library strategy: Lenti-STARR-seq

source_name: Peripheral Blood

growth_protocol: PBMC were isolated from Lymphocyte Reduction Filters obtained from male donors at Hoxworth Blood Bank at the University of Cincinnati. CD4+ T cells were isolated by magnetic negative selection using Untouched Human CD4+ Isolation Kit (Invitrogen 11346D) per manufacture’s recommendations. CD4+ T cells are cultured in Optimizer CTS (Gibco #A1048501) with 2mM L-Glutamine (Sigma # G7513) and 1x Penicillin-Streptomycin (Sigma # P4333) in incubators maintained at 37°C with 5% CO2.

extract_protocol:
HEK-293T Cells were grown in 15cm2 dishes in DMEM (Gibco #11995-065) with 10% FBS (R&D Systems #S11150). Cells were grown to 70% confluency, then DMEM was replaced two hours before transfection. Each 15cm2 plate is transfected with a total of 21ug plasmid DNA (1:2:1 ratio psPAX2-D64V:pATAC-STARR:pLAI-Env) [psPAX-D64V (Addgene #63586), and pLAI-HIV (Addgene #133996)]. DNA was mixed with 4.25x PEI (Polysciences #23966) in at total of 2mL DPBS per plate, let rest for complex formation for 10 minutes at 22°C, then added dropwise to the cell culture while swirling. 10 hours after transfection the  cells were washed once with DPBS, then cultured in DMEM with 10% FBS and 2mM Caffeine (Millipore #205548). 48 hours after transfection the media was collected and stored at 4°C. 72 hours after transfection the media is collected and pooled. The viral supernatant was sterile filtered through a 0.45uM PES filter, and loaded into Ultracentrifuge Tubes (Beckman #344058), and spun for 2.5 hours, 4°C, at 35,000g. Each pellet was dissolved in 200uL DPBS, which was left overnight at 4°C to resuspend. Concentrated Lentivirus was filtered through 0.45uM PES filter, and snap frozen in an ethanol/dry-ice bath at -80°C until use.
Lenti-STARR Transduction: Freshly isolated hCD4 T Cells were suspended to 6.6E6 cells per mL in CTS Optimizer, then combined with 0.33mL concentrated lentivirus per mL cells. Cells were spinfected in low-bind 6-well plates (Corning #3471) with 7ug/mL polybrene (MilliporeSigma #TR1003G) at 950g, 32°C, for 2 hours. Immediately following spinfection, cells were gently resuspended and cultured for 2 hours at 37°C, 5% CO2. Cells were collected and centrifuged and suspended cultured for 24 hours. Cells are treated with 100U DNaseI (NEB M0303S) for 30 minutes at 37°C.
Total RNA was purified from transduced cells (Qiagen #75144) and concentrated 7.5x using RNA Cleanup (NEB #T2040L). RNA was Reverse Transcribed according to manufacturer guidelines for STARR-Seq transcript specific primers (Supplemental File 1) with SuperScript IV (Invitrogen #18090010), using twice the recommended RNA input. cDNA was purified using a Reaction Cleanup Kit (Qiagen #28206). cDNA was amplified using a custom STARR-Seq transcript specific primer (Supplemental File 1) as previously described.2 Diluted Lenti-STARR-Seq plasmid ‘input’ control was amplified using an input concentration which yielded the same amplification cycle number as the STARR library for hCD4+ Donor I. PCR amplified library was purified using PCR cleanup columns (Qiagen #28206), and quantified for PE-150bp sequencing (Novogene).

treatment_protocol: STARR-Seq is preformed according to ATAC-STARR-Seq.2 Briefly, OMNI ATAC-Seq was performed as previously described with 160,000 human CD4+ cells (in total), in batches of 100,000 cells per reaction.3 ATAC-Seq reaction products were cleaned up using the Qiagen Reaction Cleanup Kit (Qiagen #28206) and eluted in 12uL per 100K cells transposed. Each resultant elution was amplified in 50ul with custom primers (Supplemental File 1) for 10 total cycles, as described previously.2 The amplified library was purified using a PCR Cleanup Kit (Qiagen #28104), and size selected for 150-500bp fragments on a 2% agarose gel, using 300mg per column (NEB #T1020L). The eluted ATAC-Seq library was cloned into AgeI and SalI digested pLenti-STARR using a 3:1 molar ratio (insert:backbone) in a total reaction volume of 100uL (NEB #E5520S). The reaction was concentrated to 20uL in a Reaction Cleanup (Qiagen #28206). The cloned ATAC-Lenti-STARR was electroporated into MegaX DH10B cells (Invitrogen #C6400003) using 10uL library per 100uL cells 2.0 kV, 200 Ω, 25 µF, in 20uL electroporation reactions in 0.1cm cuvettes (BioRad #165-2089) on a Harvard Apparatus (ECM Model 630). 950uL prewarmed recovery media was added and grown for one hour at 37°C. All electroporation reactions were pooled to 1L sterile Luria Broth (Fisher #BP1426), and grown in 0.2L per 2L flask, for 8hrs at 37°C, 300RPM. Each 200mL of culture was purified in EndoFree MaxiPrep Reactions (Zymo #D4203) and eluted in 0.4mL per column.

value: Peripheral Blood

tag: sample type

value: Donor IV

tag: protocol

value: STARR-Seq (ATAC)


title: bacteria+macrophage at t=285min plankt.; rep3

description: sample has been re-sequenced

tag: bacterial strain

value: THP-1 derived macrophages


title: High-fat diet fed WT mice, shCon, Tumor, rep3

description:
HFD-NC-3
Partek_HFMetYap_Quantify_to_annotation_model_(Partek_E_M)_Gene_counts.txt

tag: cell line

value: shControl

value: High-fat diet, shCon, liver metastasis


title: B16_Tumor_aPDL1_DT_CD8_4

value: Intravenous (i.v.) staining-negative, lineage-negative (CD11b, B220, NK1.1 and Ter119), TCRbeta+ and CD4- CD8+ T cells

value: 10 weeks post bone marrow transplantation

value: female


title: hCD4 STARR Donor III _2

value: Donor III


title: High-fat diet fed WT mice, shCon, Tumor, rep1

description:
HFD-NC-1
Partek_HFMetYap_Quantify_to_annotation_model_(Partek_E_M)_Gene_counts.txt

value: Liver Tumor

value: CRC liver metastasis


title: B16_Tumor_aPDL1_DT_CD8_3

value: anti-PD-L1 mAbs with DT


title: bacteria+macrophage at t=265min; rep3

description: sample has been re-sequenced

tag: genotype (bacteria)

value: Wild type

value: ATCC TIB-202


title: bacteria+macrophage at t=265min plankt.; rep3

description: sample has been re-sequenced

tag: human cell line


title: B16_Tumor_aPDL1_DT_CD8_2

value: Bone marrow chimera of Fscn1 KI


title: bacteria+macrophage at t=195min; rep3

description: sample has been re-sequenced

tag: human cell type


title: bacteria+macrophage at t=235min; rep3

description: sample has been re-sequenced


title: High-fat diet fed WT mice, shCon, Tumor, rep2

description:
HFD-NC-2
Partek_HFMetYap_Quantify_to_annotation_model_(Partek_E_M)_Gene_counts.txt

tag: tissue


title: B16_Tumor_aCD4_UT_CD8_5

value: anti-CD4 mAbs

tag: cell type

value: B16F10 melanoma


title: High-fat diet fed WT mice, Tumor, rep5

description:
HT5
Partek_LFHFMet_Quantify_to_annotation_model_(Partek_E_M)_Gene_counts.txt

value: WT

value: High-fat diet, liver metastasis


title: bacteria+macrophage at t=120min; rep3

description: sample has been re-sequenced

value: THP-1 derived macrophages


title: B16_Tumor_aCD4_UT_CD8_4

tag: strain


title: High-fat diet fed WT mice, Tumor, rep4

description:
HT4
Partek_LFHFMet_Quantify_to_annotation_model_(Partek_E_M)_Gene_counts.txt


title: B16_Tumor_aPDL1_DT_CD8_1

description:
The Fscn1 knockin (Fscn1-KI) mouse (Accession No. CDB0154E:  http://www2.clst.riken.jp/arg/mutant%20mice%20list.html) was generated by CRISPR/Cas9-mediated knockin in zygotes as previously described (Abe et al., 2020). For the homologous recombination-mediated knockin, the donor vector consisting of homology arms and P2A-DTR-P2A-Cre (insert gene) was generated to insert the P2A-DTR-P2A-Cre cassette at the 3-base upstream of the PAM sequence. For microinjection, the mixture of crRNA (CRISPR RNA) (50ng/ul), tracrRNA (trans-activating crRNA) (100ng/ul), donor vector (10ng/ul) and Cas9 protein (100ng/ul) were injected into the pronucleus of one-cell stage zygote. From 233 zygotes, 57 F0 mice were obtained and 3 of them were knockin mice confirmed by PCR, in which the PCR fragments of KI allele with 5’FW and 5’REV primers (1301 bp), and 3’FW and 3’REV primers (587 bp) were detected. The germline transmissions of knockin F0 mice were confirmed by genotyping of F1 mice. gRNA target sequences and primers used for genotyping are shown in Supplementary Table 1.
Recipient B6 mice were lethally irradiated (8.5 Gy, split into 2 doses given 3 hours apart) the day before transplantation with 3 × 106 Fscn1-KI BM cells. Mice were used for the experiments at least 8 weeks after transplantation.

tag: age


title: bacteria+macrophage at t=285min; rep2

description: sample has been re-sequenced


title: High-fat diet fed WT mice, Tumor, rep3

description:
HT3
Partek_LFHFMet_Quantify_to_annotation_model_(Partek_E_M)_Gene_counts.txt

tag: genotype


title: High-fat diet fed WT mice, Tumor, rep2

description:
HT2
Partek_LFHFMet_Quantify_to_annotation_model_(Partek_E_M)_Gene_counts.txt

tag: treatment


title: B16_Tumor_aCD4_UT_CD8_3

tag: Sex


title: High-fat diet fed WT mice, Tumor, rep1

description:
HT1
Partek_LFHFMet_Quantify_to_annotation_model_(Partek_E_M)_Gene_counts.txt


title: bacteria+macrophage at t=285min plankt.; rep2

description: sample has been re-sequenced


title: Mini-bulk samples on plate LCE516 and LCE531 indexed with RPI-16

city: Parkville

first: Peter

last: Hickey

address: 1G Royal Parade

country: Australia

institute: WEHI

data_processing:
gene and ERCC count matrix generated using scPipe 1.10.0
reads were aligned using Rsubread 2.2.6
All code used to perform data processing and analysis of the RNA-seq data are available from https://github.com/WEHISCORE/C094_Pellicci
Genome_build: GRCh38.p12
Supplementary_files_format_and_content: CSV, gene count matrices, rows represent genes and each column is a well, values corresponding to UMI counts
Supplementary_files_format_and_content: CSV, ERCC count matrices, rows represent ERCC spike-in sequences and each column is a well, values corresponding to UMI counts
Supplementary_files_format_and_content: CSV, sample sheets, rows represent a well (single-cell or mini-bulk sample) and columns contain metadata on the sample in the well
Supplementary_files_format_and_content: CSV, FACS matrix, rows represent FACS markers and each column is a (single-cell) sample, values corresponding to mean fluorescence intensities (MFI)

molecule: polyA RNA

source_name: Vg9Vd2 T cells from the thymus and peripheral blood

extract_protocol:
Neonatal and paediatric thymus and blood samples were taken from consented cardiac surgery patients (HREC38192) and obtained from the Melbourne Children’s Heart Tissue Bank (MCHTB) for this analysis. Peripheral blood mononuclear cells were isolated by a Ficoll-Paque density gradient centrifugation (supplier). Donor thymi were cut into small piece and passed through a 70mM into PBS containing 2% fetal calf serum (FACS buffer and washed x 2), prior to magnetic bead enrichment. Single-cell suspensions of human thymus were enriched for Vg9Vd2+ T cells by staining for Vg9-BV421 or Vg9-APC antibody followed by Vd2-PECy7 antibody or Vd2-PE antibody. Magnetic bead enrichment proceeded using anti-PE microbeads and passage over LS columns as per manufacturer's instructions (Miltenyl Biotec).
Single cell transcriptome libraries were generated using the CelSeq2 protocol (Hashimshony et al., 2016 Genome Biol.) with the following adaptations: Second strand synthesis was performed using NEBNext Second Strand Synthesis module (NEB, #E6111S) in a final reaction volume of 8 µl and NucleoMag NGS Clean-up and Size select magnetic beads (Macherey-Nagel, 7449970.5) were used for all DNA purification and size selection steps.

value: Vg9Vd2 T cells


title: Mini-bulk samples on plate LCE516 and LCE531 indexed with RPI-15


title: B16_Tumor_aCD4_UT_CD8_2

tag: tumour cell line


title: Low-fat diet fed WT mice, Tumor, rep5

description:
LT5
Partek_LFHFMet_Quantify_to_annotation_model_(Partek_E_M)_Gene_counts.txt

value: Low-fat diet, liver metastasis


title: bacteria+macrophage at t=265min; rep2

description: sample has been re-sequenced


title: B16_Tumor_aCD4_UT_CD8_1

value: Intravenous (i.v.) staining-negative, lineage-negative (CD11b, B220, NK1.1 and Ter119), TCRbeta+ and CD4- CD8+ T cells

tag: organ


title: Mini-bulk samples on plate LCE515 indexed with RPI-12


title: bacteria+macrophage at t=265min plankt.; rep2

description: sample has been re-sequenced


title: Low-fat diet fed WT mice, Tumor, rep4

description:
LT4
Partek_LFHFMet_Quantify_to_annotation_model_(Partek_E_M)_Gene_counts.txt


title: Low-fat diet fed WT mice, Tumor, rep3

description:
LT3
Partek_LFHFMet_Quantify_to_annotation_model_(Partek_E_M)_Gene_counts.txt


title: B16_Tumor_aCD4_DT_CD8_3

value: anti-CD4 mAbs with DT

value: 10 weeks post bone marrow transplantation

tag: date


title: Single cells on plate LCE513 indexed with RPI-9

source_name: Vg9Vd2 T cells from the thymus and peripheral blood; SKW3 control cells

value: Vg9Vd2 T cells; SKW3 control cells


title: B16_Tumor_aCD4_DT_CD8_4

value: female


title: Single cells on plate LCE514 indexed with RPI-10


title: bacteria+macrophage at t=235min; rep2

description: sample has been re-sequenced


title: Low-fat diet fed WT mice, Tumor, rep2

description:
LT2
Partek_LFHFMet_Quantify_to_annotation_model_(Partek_E_M)_Gene_counts.txt

value: Liver Tumor

value: CRC liver metastasis


title: bacteria+macrophage at t=195min; rep2

description: sample has been re-sequenced

value: Wild type


title: Low-fat diet fed WT mice, Tumor, rep1

description:
LT1
Partek_LFHFMet_Quantify_to_annotation_model_(Partek_E_M)_Gene_counts.txt


title: Single cells on plate LCE511 indexed with RPI-3


title: bacteria+macrophage at t=120min; rep2

description: sample has been re-sequenced

value: ATCC TIB-202


title: bacteria+macrophage at t=285min; rep1

description: sample has been re-sequenced


title: Single cells on plate LCE512 indexed with RPI-5


title: bacteria only at t=30min; rep4

organism: Vibrio cholerae O1 biovar El Tor

source_name: whole bacterial cells


title: B16_Tumor_CD4PDL1_UT_CD8_4

value: Bone marrow chimera of Fscn1 KI

value: combination of anti-CD4 and anti-PD-L1 mAbs


title: B16_Tumor_aCD4_DT_CD8_1

value: B16F10 melanoma


title: B16_Tumor_aCD4_DT_CD8_2


title: bacteria+macrophage at t=285min plankt.; rep1

description: sample has been re-sequenced


title: bacteria only at t=30min; rep3


title: bacteria+macrophage at t=265min; rep1

description: sample has been re-sequenced


title: B16_Tumor_CD4PDL1_UT_CD8_2


title: Single cells on plate LCE508 indexed with RPI-1


title: Single cells on plate LCE509 indexed with RPI-2


title: B16_Tumor_CD4PDL1_UT_CD8_3


title: 8h C U 15

city: Solna

first: susanne

last: neumann

address: Solnavägen 1

country: Sweden

institute: Karolinska Instituet

description:
P9913_210
DTM: H8_C_N
Experiment: H202

data_processing:
Clustering was done by 'cBot' and samples were sequenced on HiSeq2500 (HiSeq Control Software 2.2.58/RTA 1.18.64) with a 2x126 setup using 'HiSeq SBS Kit v4' chemistry. The Bcl to FastQ conversion was performed using bcl2fastq_v2.19.1.403 from the CASAVA software suite. The quality scale used is Sanger / phred33 / Illumina 1.8+.
Data mapped with nfcore pipeline nf-core RNAseq version-1.0.
Assembly: Mus musculus, GRCm38
Supplementary files format and content: *Aligned.sortedByCoord.out.gene_abund.txt: Tab-delimited text files include information regarding Gene ID, Gene name and position, FPKM and TPM values of each gene analysed for each sample.
Supplementary files format and content: RNAseq_SVZ_controls.xlsx: Excel file includes information regarding Gene ID, Gene name and position, FPKM and TPM values of each gene analysed for each sample.
Supplementary files format and content: GenecountsFiltered20230509.txt: Tab-delimited text file includes DESeq2 normalized counts for every sample.

source_name: NPC

growth_protocol: Primary neural precursor cells were isolated from adult female C57BL/6 mice, 5–8 weeks old, according to a modified protocol previously described by Johansson et al. [johansson CB, Momma S, Clarke DL, Risling M, Lendahl U and Frisen J. (1999). Identification of a neural stem cell in the adult mammalian central nervous system. Cell 96:25–34. Crossref, Medline, Google Scholar].

extract_protocol:
RNeasy Kit according to manufacturer's protocol.
Illumina TruSeq Stranded mRNA, Poly-A selection, default, 1527, 3 lanes minimum 14 mio reads.

treatment_protocol: NPCs were exposed to H2O2 at 100 μM for 1, 8, and 24 h; washed; and collected or cultured on PDL coated plates for the indicated amount of time without H2O2 as controls. Total mRNA was isolated at these different time points during the experiment.

value: Female

value: 5-8 weeks

value: NPC

value: No mutation

value: 8h sham exposure PBS


title: bacteria only at t=30min; rep2


title: bacteria+macrophage at t=265min plankt.; rep1

description: sample has been re-sequenced

value: THP-1 derived macrophages


title: B16_Tumor_CD4PDL1_UT_CD8_1

value: Intravenous (i.v.) staining-negative, lineage-negative (CD11b, B220, NK1.1 and Ter119), TCRbeta+ and CD4- CD8+ T cells


title: 8h C U 13

description:
P9913_209
DTM: H8_C_N
Experiment: H202


title: bacteria only at t=0min; rep3


title: bacteria+macrophage at t=235min; rep1

description: sample has been re-sequenced


title: 8h C U 14

description:
P9913_118
DTM: H8_C_N
Experiment: H202


title: bacteria+macrophage at t=195min; rep1

description: sample has been re-sequenced


title: bacteria only at t=30min; rep1


title: 1h C U 2

description:
P9913_102
DTM: H1_C_N
Experiment: H202

value: 1h sham exposure PBS


title: B16_Tumor_CD4PDL1_DT_CD8_3

value: combination of anti-CD4 and anti-PD-L1 mAbs with DT

value: 10 weeks post bone marrow transplantation


title: B16_Tumor_CD4PDL1_DT_CD8_4

value: female


title: bacteria only at t=0min; rep2


title: B16_Tumor_CD4PDL1_DT_CD8_2


title: 1h C U 1

description:
P9913_101
DTM: H1_C_N
Experiment: H202


title: bacteria+macrophage at t=120min; rep1

description: sample has been re-sequenced


title: bacteria only at t=0min; rep1


title: heads_1DONP_nonSizeSelected_6

status: Public on May 10 2023

city: Freiburg

first: Valérie

last: Hilgers

address: Stübeweg 51

institute: MPI of Immunobiology and Epigenetics

description: CIA assembly

data_processing:
instrument model: MinION 1B
CIA assembly workflow: FLAIR (Tang et al., 2020) was used for transcriptome assembly. Samples were pooled to perform the assembly. For FLAIR collapse, the Eukaryotic Promoter Database EPD (Dreos et al., 2017) was provided to retain only reads with a supported TSS at their 5' end, using --max_ends 5 to allow for multiple 5’-3’ end identification. A minimum of three full-length reads were required for an isoform to be collapsed in the assembly.  Assemblies were filtered for 3' ends that likely originated from internal priming or truncation during library preparation. We used FLAM-seq and RNA Direct data, as both of these methods allow for poly(A) tail detection, to perform poly(A) tail calling. Only reads containing a poly(A) tail were retained, and were trimmed to a single nucleotide preceding the poly(A) tail. Single nucleotide reads were clustered in 20-nt windows; clusters supported by at least two reads were included in the PAS database. The following filtering parameters were considered: 1) All isoforms overlapping a 3' end in a window of 100 nt were retained, 2) 3' ends found in the assembly more distal than the 3' ends found in the database were retained only if they were within the reference annotation and contained a AATAAA signal. For 3' end Correction: 1) 3’ UTR bins were created using the PAS database, starting from the end of the open reading frame, between each consecutive PAS, to the most distal PAS. Isoform 3’ ends falling within the last bin of the 3’ UTR (between the two distal-most PASs) were corrected to the most distal bin, provided the isoform covered more than 10% of the last bin. After assembling of all tissues and methods transcriptomes were merged into a single gtf file using TAMA merge. Here reported 59K isoforms in CIA assembly.allTissues59K.gtf file.
Assembly: D. melanogaster Assembly: dm6_ensembl ; D. melanogaster Annotation: ensembl release 96
Supplementary files format and content: w1118_Heads_1DONP_assembly.gtf: Gene transfer format file containing the integration of long read sequencing assemblies of the Head tissue: Processed gtf (General Transfer Format) output of the FLAIR assembly, each row represents an exon. Columns are: chromosome name, source, feature type, start,end,score,strand, In the 9th column is metadata assigned to each exon: gene_id, transcript_id, exon number.
Supplementary files format and content: w1118_embryo14_16hrs_embryo18_20hrs_1DONP_assembly.gtf: Gene transfer format file containing the integration of long read sequencing assemblies of the Embryo time points: Processed gtf (General Transfer Format) output of the FLAIR assembly, each row represents an exon. Columns are: chromosome name, source, feature type, start,end,score,strand, In the 9th column is metadata assigned to each exon: gene_id, transcript_id, exon number.
Supplementary files format and content: w1118_Ovaries_1DONP_assembly.gtf: Gene transfer format file containing the integration of long read sequencing assemblies of the Ovary tissue: Processed gtf (General Transfer Format) output of the FLAIR assembly, each row represents an exon. Columns are: chromosome name, source, feature type, start,end,score,strand, In the 9th column is metadata assigned to each exon: gene_id, transcript_id, exon number.

organism: Drosophila melanogaster

source_name: heads

growth_protocol: Flies were grown in 25C on standard apple juice based food

extract_protocol:
For head transcriptomes, 3-day-old w1118 flies were collected and flash-frozen in liquid nitrogen and heads were used for RNA extraction. For ovary transcriptomes, 3 day old w1118 virgin females were collected and dissected using 20 ovaries per replicate. For embryo transcriptomes, eggs from w1118 flies were collected for two hours on agar plates and aged for either 14h (14-16h AEL embryos) or 18h (18-20h AEL) at 25°C. 50 embryos were used for each replicate.
For generation of full-length cDNA libraries, polyadenylated RNA molecules were isolated from total RNA preparations using the NEB’s NEBNext® Poly(A) mRNA Magnetic Isolation Module (E7490, NEB). Purified polyadenylated RNA molecules were used for nanopore library preparation using the cDNA-PCR Sequencing protocol (SQK-PCS109, Oxford Nanopore was used). The following modifications were made to the procedure. To eliminate short reads from the final data both input polyadenylated RNA molecules and cDNA molecules were cleaned upon further processing using AMPure XP beads (A63881, Beckman&Coulter) using a magnetic bead sample ratio of 0.4. To exclusively retain cDNA fragments > 3kb the BluePippin device and appropriate separation DNA gel cassettes were used (sage science). Finally, the cDNA was amplified using 14 PCR cycles and 12 min extension time at 65°C. Libraries were sequenced on a MinION 1B or GridION sequencing device from Oxford Nanopore Technologies. Reads were processed using the pipeline described in: git@github.com:LeilyR/nanoporeReads_dataTransfer.git (For cDNA data: guppy-5.0.7 model: dna_r9.4.1_450bps_sup.cfg was used).
cDNA-PCR Sequencing protocol (SQK-PCS109)

treatment_protocol: untreated

value: heads

tag: developmental stage

value: adults

value: wild type (w1118)


title: B16_Tumor_CD4PDL1_DT_CD8_1

value: Bone marrow chimera of Fscn1 KI


title: bacteria only at t=0min; rep4

value: Wild type


title: bacteria+macrophage mixed sample

data_processing:
Read-1 files were mapped to the hg19 genome assembly using Bowtie2 (version 2.3.4.1)
Demultiplexing and reads counting were based on 7bp barcodes for each sample from Read-2, and performed using the pipeline described in Jaitin et al. 2014
Data were normalized to a library size factor,calculated by dividing the total number of reads from each sample to the median of the total number of reads across all samples. And log transformed
A minimal expression threshold was set to 3.5.
Genome_build: hg19
Supplementary_files_format_and_content: Expression data sets: csv files include reads counts before and after normalization.host_raw_count_data.csv - raw counts from each sample, host_final_normalized_data.csv - normalized, log2 transformed, with minimal value set to 5, annotation.txt - file contains the pool barcodes per each sample  for MARSseq pipeline (Jaitin et al. 2014).
Genome_build: hg19
Supplementary_files_format_and_content: macrophage_significantly_up_and_downregulated_genes.xlsx: Excel file includes differentially expressed genes in human macrophages: numbers in each row indicate the log2 fold change calculated for each sample from an average of all naive samples (macrophage only).

growth_protocol: Vibrio cholerae C6706 WT, ∆mshA∆tcpA, ∆hlyA or ∆mshA∆tcpA∆hlyA was exposed to THP-1 derived macrophages in a well of a 12-well plate for a defined time at 37°C and 5% CO2.

extract_protocol:
RNA was isolated using the mirVana Kit(Ambion, cat. no. AM1560) according to the manufacturer`s protocol. DNA was digested and the remaining RNA was purified via ethanol precipitation. The RNA integrity was checked using the Agilent 1450 TapeStation.
RNA-seq libraries were prepared according to MARS-seq libraries protocol (Jaitin et al Science 2014)

value: mixed sample

value: ATCC TIB-202


title: Ovaries_1DONP_2

description: CIA assembly

source_name: Ovaries

value: Ovaries


title: Ovaries_1DONP_3

description: CIA assembly


title: Ovaries_1DONP_1

description: CIA assembly


title: embryo18_20hrs_1DONP_1

description: CIA assembly

source_name: embryos 18-20 hrs

value: embryos 18-20 hrs

value: embryos


title: embryo18_20hrs_1DONP_2

description: CIA assembly


title: embryo18_20hrs_1DONP_3

description: CIA assembly


title: heads_1DONP_3

description: CIA assembly


title: heads_1DONP_4

description: CIA assembly


title: embryo14_16hrs_1DONP_2

description: CIA assembly

source_name: embryos 14-16 hrs

value: embryos 14-16 hrs


title: heads_1DONP_2

description: CIA assembly


title: heads_1DONP_1

description: CIA assembly


title: embryo14_16hrs_1DONP_1

description: CIA assembly


title: ovaries_RNADirect_3

description:
polyA tail length database, CIA assembly
w1118_ovaries_onp.rds.assembly.gtf,ciaPolyADatabase.tsv

data_processing:
instrument model: MinION 1B
For RNA direct datasets, poly(A) tail length estimations were performed using https://github.com/nanoporetech/pipeline-polya-ng. Lengths were summarized at gene level as the median poly(A) tail length per gene. At transcript level, tails were assigned to transcripts and summarized as median poly(A) tail length per transcript.
CIA assembly workflow: FLAIR (Tang et al., 2020) was used for transcriptome assembly. Samples were pooled to perform the assembly. For FLAIR collapse, the Eukaryotic Promoter Database EPD (Dreos et al., 2017) was provided to retain only reads with a supported TSS at their 5' end, using --max_ends 5 to allow for multiple 5’-3’ end identification. A minimum of three full-length reads were required for an isoform to be collapsed in the assembly.  Assemblies were filtered for 3' ends that likely originated from internal priming or truncation during library preparation. We used FLAM-seq and RNA Direct data, as both of these methods allow for poly(A) tail detection, to perform poly(A) tail calling. Only reads containing a poly(A) tail were retained, and were trimmed to a single nucleotide preceding the poly(A) tail. Single nucleotide reads were clustered in 20-nt windows; clusters supported by at least two reads were included in the PAS database. The following filtering parameters were considered: 1) All isoforms overlapping a 3' end in a window of 100 nt were retained, 2) 3' ends found in the assembly more distal than the 3' ends found in the database were retained only if they were within the reference annotation and contained a AATAAA signal. For 3' end Correction: 1) 3’ UTR bins were created using the PAS database, starting from the end of the open reading frame, between each consecutive PAS, to the most distal PAS. Isoform 3’ ends falling within the last bin of the 3’ UTR (between the two distal-most PASs) were corrected to the most distal bin, provided the isoform covered more than 10% of the last bin.
Assembly: D. melanogaster Assembly: dm6_ensembl ; D. melanogaster Annotation: ensembl release 96
Supplementary files format and content: Processed gtf (General Transfer Format) output of FLAIR assembly, each row represents an exon. Columns are: chromosome name, source, feature type, start,end,score,strand, In the 9th column metadata assigned to each exon: gene_id, transcript_id, exon number
Supplementary files format and content: tsv file: table in which each row represents a poly(A) tail read. Columns: CIATRANSID: CIA transcript assignment, polya_length: tail length estimate, sample_name: sample source,tissue:tissue, gene_id: flybase gene id, symbol: flybase gene name, ensemblTRANSID: transcript id name in ensembl database if "novel" only found in CIA transcriptome, strand: transcript strand, transcript_length: length of the transcript, coding: coding potential of transcription, source: tissue source
Library strategy: Direct RNA sequencing (DRS)

source_name: ovaries

extract_protocol:
For head transcriptomes, 3-day-old w1118 flies were collected and flash-frozen in liquid nitrogen and heads were used for RNA extraction. For ovary transcriptomes, 3 day old w1118 virgin females were collected and dissected using 20 ovaries per replicate. For embryo transcriptomes, eggs from w1118 flies were collected for two hours on agar plates and aged for either 14h (14-16h AEL embryos) or 18h (18-20h AEL) at 25°C. 50 embryos were used for each replicate.
For direct RNA sequencing, polyadenylated RNA molecules were isolated from total RNA preparations using the Dynabeads™ mRNA Purification Kit (61006, Invitrogen). Multiple poly-A+ pulldowns were pooled to reach 500 ng PolyA+ input for library preparation using the Direct RNA sequencing kit (SQK-RNA002, Oxford Nanopore Technologies). Libraries were sequenced on a MinION 1B or GridION sequencing device from Oxford Nanopore Technologies. Reads were processed using the pipeline described in: git@github.com:LeilyR/nanoporeReads_dataTransfer.git? (For RNA direct data: guppy-5.0.7 model: rna_r9.4.1_70bps_hac.cfg was used).  git@github.com:LeilyR/nanoporeReads_dataTransfer.git? (For cDNA data: guppy-5.0.7 model: dna_r9.4.1_450bps_sup.cfg was used).
Direct RNA sequencing kit (SQK-RNA002)

value: ovaries


title: ovaries_RNADirect_2


title: embryo14_16hrs_RNADirect_2

description:
polyA tail length database, CIA assembly
w1118_embryos_onp.rds.assembly.gtf,ciaPolyADatabase.tsv


title: embryo14_16hrs_RNADirect_3


title: Wild-type mouse ES cells on tissue-culture plastic (2iLIF), bulk RNA-seq, replicate 3

city: Cambridge

first: Florian

last: Hollfelder

address: 80 tennis court road

country: United Kingdom

institute: University of Cambridge

description:
Mouse ES cells were cultured on tissue-culture plastic in 2iLIF conditions. Libraries were prepared  using the Smart-seq2 methodology, on a 1,000 cells. The experiments were performed in triplicates, this sample is triplicate 3.
mES_3d_gels_versus_tcp_2iLIF.csv

data_processing:
FastQC quality control
For single-cell, de-multiplexing using Pheniqs
For bulk RNA-seq experiments, alignment to the genome using the STAR aligner. For single-cell data, processing into gene expression matrices using zUMIs
For bulk RNA-seq experiments, quantification of events using featureCounts.
For bulk RNA-seq experiments, downstream processing using DESeq2. For single-cell experiments, downstream processing using Scanpy and scVelo.
Genome_build: For bulk RNA-seq experiments, mm10. For single-cell experiments GRCm38
Supplementary_files_format_and_content: For bulk RNA-seq experiments, the processed data files are normalized gene expression counts. For single-cell data, the processed data files are UMI counts for both introns and exons after pre-processing fwith the zUMIs pipeline

source_name: tissue culture

growth_protocol: Cultured in 2iLIF

extract_protocol:
Smart-seq2 protocol on a ~1,000 cells
Smart-seq2

treatment_protocol: Washed twice with PBS

value: mouse embryonic stem cells

tag: agent

value: cultured in 2iLIF

tag: library strategy


title: heads_RNADirect_1

description:
polyA tail length database, CIA assembly
w1118_heads_onp.rds.assembly.gtf,ciaPolyADatabase.tsv


title: Wild-type mouse ES cells on tissue-culture plastic (2iLIF), bulk RNA-seq, replicate 1

description:
Mouse ES cells were cultured on tissue-culture plastic in 2iLIF conditions. Libraries were prepared using the Smart-seq2 methodology, on a 1,000 cells. The experiments were performed in triplicates, this sample is triplicate 1.
mES_3d_gels_versus_tcp_2iLIF.csv


title: Wild-type mouse ES cells in gels (2iLIF), bulk RNA-seq, replicate 2

description:
Mouse ES cells were encapsualted in agarose microgels and cultured in 2iLIF conditions. Libraries were prepared on the encapsulated cells using the Smart-seq2 methodology, on a 1,000 cells. The experiments were performed in triplicates, this sample is triplicate 2.
mES_3d_gels_versus_tcp_2iLIF.csv

growth_protocol: Agarose microgels embedding, cultured in 2iLIF


title: Wild-type mouse ES cells in gels (2iLIF), bulk RNA-seq, replicate 3

description:
Mouse ES cells were encapsualted in agarose microgels and cultured in 2iLIF conditions. Libraries were prepared on the encapsulated cells using the Smart-seq2 methodology, on a 1,000 cells. The experiments were performed in triplicates, this sample is triplicate 3.
mES_3d_gels_versus_tcp_2iLIF.csv


title: embryo14_16hrs_RNADirect_1


title: Wild-type mouse ES cells on tissue-culture plastic (2iLIF), bulk RNA-seq, replicate 2

description:
Mouse ES cells were cultured on tissue-culture plastic in 2iLIF conditions. Libraries were prepared using the Smart-seq2 methodology, on a 1,000 cells. The experiments were performed in triplicates, this sample is triplicate 3.
mES_3d_gels_versus_tcp_2iLIF.csv


title: embryo14_16hrs_1DONP_3

description: CIA assembly


title: heads_1DONP_nonSizeSelected_5

description: CIA assembly


title: HMZ 1030 3

city: Riverside

first: Frances

last: Sladek

address: 900 University Avenue

institute: University of California

library_source: genomic

data_processing:
Basecalls were performed using bcl2fastq v2.17 for Novaseq output.
ChIP-seq reads were aligned to the mouse reference genome, version mm10, with Bowtie2 using the default parameters
Peaks were called using MACS2  using default parameters for individual samples, as well as a pooled peak dataset using the SPMR parameter using the
Aligned reads and MACS2 peak-sets were analyzed with DiffBind to identify common and uniquely bound regions of the genome
bigWig files were generated using the genomicRanges R package. Score represents the normalized coverage of DNA fragments at a given genomic coordinate. narrowPeak files were generated using MACS2 with default settings.
Assembly: mm10
Supplementary files format and content: bigWig, narrowPeak

molecule: genomic DNA

source_name: Liver

growth_protocol: Livers from male mice on standart vivarium chow diet

extract_protocol:
Fresh tissue was fixed with 1% formaldehyde for 15 minutes. After quenching and cell lysis, chromatin was sonicated using a Sonic Dismembtrator 500 (Fisher Scientific). DNA fragments with 200-500 bps were selected
ChIP-seq libraries were prepared using the BIOO Scientific ChIPseq DNA library kit. 2-22 ng of DNA was used as starting material for input and ip samples. Libraries were amplified using a thermocycler. Libraries were validated using ???

treatment_protocol: no treatment

value: male

value: exon swap a7HMZ (P2-HNF4a only)

value: 16 to 20 weeks

tag: diet

value: fed ad libitum (LabDiet, #5001)

value: mixed 129/Sv plus C57BL/6 background

value: Liver

tag: chip antibody

value: HNF4a (alpha-445)


title: HMZ 1030 2


title: ovaries_RNADirect_1

value: adults

value: wild type (w1118)


title: Wild-type mouse ES cells in gels (2iLIF), bulk RNA-seq, replicate 1

description:
Mouse ES cells were encapsualted in agarose microgels and cultured in 2iLIF conditions. Libraries were prepared on the encapsulated cells using the Smart-seq2 methodology, on a 1,000 cells. The experiments were performed in triplicates, this sample is triplicate 1.
mES_3d_gels_versus_tcp_2iLIF.csv


title: WT 1030 2

value: WT


title: WT 1030 3


title: HMZ 1030 1


title: Uterus, pregnancy, Day 4, Cfp1 cKO rep1

city: Pangyo

first: Seung Chel

last: Yang

address: Seongnam, Gyeonggi

country: South Korea

institute: CHA University

data_processing:
Subsequently, libraries were checked using the Agilent 2100 bioanalyzer (DNA High Sensitivity Kit) to evaluate the mean fragment size.
Quantification was performed via the library quantification kit using a StepOne Real-Time PCR System (Life Technologies, Inc., USA).
mRNA-Seq reads were mapped using TopHat software tool to obtain the alignment file. Differentially expressed genes were determined based on counts from unique and multiple alignments using coverage in Bedtools.
The RT (Read Count) data were processed based on the Quantile normalization method using EdgeR within R (R development Core Team, 2016) using Bioconductor. The alignment files also were used for assembling transcripts, estimating their abundances and detecting differential expression of genes or isoforms using cufflinks.
Assembly: mm10
Supplementary files format and content: Additionally, we used the FPKM (fragments per kilobase of exon per million fragments) as the method of determining the expression level of the gene regions.

source_name: Uterine cell

extract_protocol:
Total RNA was extracted from mouse uteri on day 4 of pregnancy using Trizol Reagent (Invitrogen Life Technologies, San Diego, CA, USA) according to the manufacturer’s protocols.
Libraries were prepared from 2 μg of total RNA in Cfp1f/f and Cfp1d/d mouse uterus on Day 4 using the SMARTer stranded mRNA-Seq kit (Clontech Laboratories, Inc., USA).

value: Uterine cell

value: C57BL/6 mice

value: Cfp1 cKO

value: 8–10 weeks old


title: Uterus, pregnancy, Day 4, WT rep3


title: Uterus, pregnancy, Day 4, WT rep2


title: H3k4me3 ChIP seq Cfp1 cKO input

description: Uterus, pregnancy, Day 4

data_processing:
The size of the library was assessed by Agilent 2100 bioanalyzer (Agilent Technologies, Amstelveen, The Netherlands).
High-throughput sequencing was performed as paired-end 100 sequencing using HigSeq 2500 (Illumina, Inc. USA).
Assembly: mm10
Supplementary files format and content: bigWig, narrowPeak (except for Input sample)

extract_protocol:
Chip analysis was performed with a slight modification of the manufacturer's instructions using the ChIP-IT Express Enzymatic kit (Active Motif, Carlsbad, CA, USA). Briefly, Cfp1f/f and Cfp1d/d mouse uterus horns on Day 4 were cut vertically, and then separated by epithelial cells and stromal cells except smooth muscles using a scalpel and chopping small. Afterward, cells were fixed with 1% formaldehyde, DNA fragments between 150 and 500 base pairs (bp) were obtained by enzymatic shearing cocktail.
The construction of the library was performed using NEBNext® UltraTM DNA Library Prep Kit for Illumina (New England Biolabs, UK) according to the manufacturer’s instructions. Briefly, the chipped DNA was ligated with adaptors. The library was purified by using magnetic beads to remove all reaction components.


title: WT 1030 1


title: Uterus, pregnancy, Day 4, Cfp1 cKO rep3


title: H3k4me3 ChIP seq Cfp1 cKO IP

description: Uterus, pregnancy, Day 4


title: H3k4me3 ChIP seq Cfp1 WT input

description: Uterus, pregnancy, Day 4


title: H3k4me3 ChIP seq Cfp1 WT IP

description: Uterus, pregnancy, Day 4


title: E68xin: db/db mice 2, left ventricular, 16 week, DCM, RNA-seq

city: chongqin

first: Yao

state: chongqin

address: Gaotanyan

institute: TMMU

data_processing:
Indexed libraries were sequenced in the 2×150bp configuration on the Illumina HiSEQ platform. Briefly, first strand and second strand cDNA were synthesized from the input RNA, single primer isothermal amplification (SPIA) of the resultant cDNA was performed, followed by mechanical shearing of ds-cDNA (COvaris E220 ultrasonicator; Covaris Inc, MA). Subsequently, end repair and A-tailing of sheared cDNA and construction of unique barcoded libraries by addition of adapters and PCR amplification (8 cycles) was conducted. The Agencourt AmPure XP bead (Beckmann Coulter) purified libraries were quantified by qPCR and the size distribution was checked using Agilent TapeStation 2200 system. The libraries were subjected to paired-end 150bp sequencing on HiSeq 2500 sequencing system (Illumina).
For all samples including public data, paired-end reads were mapped to the mm10 genome assembly using STAR with default parameters. Duplicate and multi-mapped reads are removed with samtools. Following alignment, reads were counted per gene and fragments per kilobase per million (FPKM) using the featureCounts tool and the GENCODE m28 and NONCODE v6 annotation.
Assembly: mm10
Supplementary files format and content: tab-delimited text files include RPKM values for each Sample

source_name: left ventricular

growth_protocol: All the procedures were performed under the guidelines for the animal care, which is approved by the Animal Care and Ethics Committee of Southwest Jiaotong University. Two murine strains with C57BL/6 background including db/m and db/db mice are used as the spontaneous diabetic cardiomyopathy model and healthy control model, respectively. Three newborn healthy male db/m mice and three newborn healthy male db/db mice were purchased from Shanghai Nanfang Model Biotechnology Co. LTD (Shanghai, China).

extract_protocol:
RNA was isolated from the left ventricular of three db/db mice and three db/m mice, respectively.
RNA was used to generate barcoded cDNA libraries using the NEBNext Ultra RNA library prep with rRNA depletion (New England Biolabs).

treatment_protocol:
db/m mice were given ad libitum access to tap water and laboratory feed for 16w. db/db mice were fed with a high-fat diet for 16w.
The ultrasound system (VisualSonics, Toronto, ON, Canada) was used for echocardiographic measurements. After being anesthetized with avertin, the mice were shaved to expose the chest area and then fixed onto a flat plate. Left ventricular systolic diameter (LVDs), LV diastolic diameter (LVDd), LV ejection fraction (EF), and LV fractional shortening (FS) were determined and were the average of 6 consecutive cardiac cycles

value: left ventricular

value: db/db

value: high-fat diet

tag: time


title: B2xin: db/m mice 2, left ventricular, 16 week, control, RNA-seq

value: db/m

value: laboratory feed


title: B1xin: db/m mice 1, left ventricular, 16 week, control, RNA-seq


title: B3xin: db/m mice 3, left ventricular, 16 week, control, RNA-seq


title: E69xin: db/db mice 3, left ventricular, 16 week, DCM, RNA-seq


title: m12_WD+VC rep1

city: Pittsburgh

first: Shuchang

last: Liu

state: PA

address: 203 Lothrop Street

institute: University of Pittsburgh

description: m12_WD+VC rep1

data_processing:
Low-quality reads and adapter sequences were filtered out by tool Trimmomatic (version 0.38)
Reads were aligned to mouse reference genome mm10 by HISAT2 aligner (version 2.1.0)
Gene counts were quantified by htseq-count (version 0.11.2)
Genome_build: mm10
Supplementary_files_format_and_content: htseq-count

source_name: liver

extract_protocol:
Total RNA was extracted using Trizol and treated with DNAse1. Ribosomal RNA was removed from the samples using RIBO-ZeroTM Magnetic kit (Epicentre, Madison, WI). RNA was reverse-transcribed to cDNA and amplified using TruSeq™ RNA Sample Prep Kit v2 from Illumina, Inc (San Diego, CA). The cDNA library was similarly sequenced in Illumina HiSeq2500 sequencer.
RNA libraries were prepared for sequencing using standard Illumina protocols

treatment_protocol: Mice were housed in a pathogen-free barrier accredited by the Association for Assessment and Accreditation of Laboratory Animal Care and procedures were approved by the local Institutional Animal Care and Use Committee. Food and tap water were provided ad libitum. Mice were fed either a low-fat control diet (CD, 13% fat, Teklad diets, # TD.120336) or a Western-style high-fat diet (WD, 42% fat, Teklad diets, # TD.07511) for 12 weeks. Mice were exposed to VC (Kin-tek, La Marque, TX) at ∼0.85 ± 0.1 ppm, or air, in inhalation chambers for 6 hours per day, 5 days per week for 12 weeks. Body weight and food consumption were monitored throughout the exposure regimen. Animals were sacrificed either immediately following 12 weeks of exposure or following an additional 9 months of housing (±WD feeding). At sacrifice, mice were fasted for 4 hours and anesthetized with ketamine/xylazine (100/15 mg/kg, i.p.). Blood was collected from the vena cava, and citrated plasma was stored at -80°C. Liver tissue was snap-frozen in liquid nitrogen, embedded in frozen specimen medium (Sakura Finetek, Torrance, CA), or was fixed in 10% neutral buffered formalin.

tag: model

value: 12 months

tag: gender

value: Male


title: m12_WD+VC rep2

description: m12_WD+VC rep2


title: m12_WD+VC rep3

description: m12_WD+VC rep3


title: m12_WD rep1

description: m12_WD rep1


title: m12_WD rep2

description: m12_WD rep2


title: m12_WD rep3

description: m12_WD rep3


title: m12_CD+VC rep1

description: m12_CD+VC rep1


title: m12_CD+VC rep2

description: m12_CD+VC rep2


title: Uterus, pregnancy, Day 4, Cfp1 cKO rep2

tag: cell type


title: m12_CD rep2

description: m12_CD rep2

tag: strain


title: m12_CD+VC rep3

description: m12_CD+VC rep3


title: m12_CD rep3

description: m12_CD rep3


title: Uterus, pregnancy, Day 4, WT rep1

tag: age


title: w12_WD+VC rep3

description: w12_WD+VC rep3

value: 12 weeks


title: w12_WD+VC rep2

description: w12_WD+VC rep2


title: w12_WD+VC rep1

description: w12_WD+VC rep1


title: m12_CD rep1

description: m12_CD rep1


title: w12_WD rep1

description: w12_WD rep1


title: w12_CD+VC rep2

description: w12_CD+VC rep2


title: CFP1 ChIP seq BL6-WT-PD4-IP

description: Uterus, pregnancy, Day 4

tag: genotype


title: w12_WD rep3

description: w12_WD rep3


title: CFP1 ChIP seq BL6-WT-PD4-input

description: Uterus, pregnancy, Day 4


title: E67xin: db/db mice 1, left ventricular, 16 week, DCM, RNA-seq

tag: tissue

tag: treatment


title: w12_CD rep3

description: w12_CD rep3


title: w12_CD rep2

description: w12_CD rep2


title: w12_CD+VC rep1

description: w12_CD+VC rep1


title: w12_CD rep1

description: w12_CD rep1


title: HCT116-ZIC2KO-C3-R1

city: Chicago

first: Ali

last: Shilatifard

state: IL

address: 320 E Superior St

department: Department of Biochemistry and Molecular Genetics

institute: Northwestern University Feinberg School of Medicine

data_processing:
Basecalls were performed using bcl2fastq v2.17 for NextSeq output.
Both ChIP-seq and RNA-seq reads were trimmed from 3' end until the final base had a quality score > 30, using Trimmomatic v0.33, discarding reads left with < 20 bp
ChIP-seq reads were aligned to UCSC mm9 using Bowtie version 0.12.9. Only uniquely mapping reads with at most two mismatches were retained.
RNA-seq reads were aligned to UCSC mm9 using Tophat version 2.0.9. Only uniquely mapping reads with at most two mismatches were retained.
To create ChIP-seq coverage plots, the locations of the mapped ChIP-seq reads were extended to 150 bp to represent sequenced fragments, renormalized (to reads per million, rpm) and reformatted in the bigWig file format.
To create RNA-seq coverage plots, the mapped RNA-seq reads were split according to strand, renormalized (to reads per million, rpm) and reformatted in the bigWig file format.
Assembly: hg19
Supplementary files format and content: bigWig files were generated using the genomicRanges R package. Score represents the normalized coverage of RNA or DNA fragments at a given genomic coordinate.

growth_protocol: Cell medium was composed as follow: DMEM 1X (Thermo Fisher), 10% serum (Corning), 1X glutamine (Life Technologies), 1X penicillin/streptomycin (Life Technologies), cells were grown in a CO2 incubator (5% CO2) at 37˚C

extract_protocol:
RNA was extracted using QIAGEN RNeasy kit. DNA after ChIP protocol was isolated using QIAGEN PCR purification kit.
ChIP-sequencing libraries were prepared using the KAPA HTP Library Preparation Kit complemented with NEXTflex DNA Barcodes from Bioo Scientific. 10 ng of DNA was used as starting material for input and ip samples. Libraries were amplified using 13 cycles on the thermocycler. Post amplification libraries were size selected at 250- 450bp in length using Agencourt AMPure XP beads from Beckman Coulter. Libraries were validated using the Agilent High Sensitivity DNA Kit. RNA-sequencing libraries were prepared using the Illumina TruSeq Stranded Total RNA Preparation Kit with Ribo-Depletion. Input RNA quality was validated using the Agilent RNA 6000 Nano Kit. 200ng-1 µg of total RNA was used as starting material. Libraries were validated using the Agilent DNA 1000 Kit

tag: cell line

value: colorectal cancer cells

tag: passages

value: Low passages (6-10)


title: HCT116-ZIC2KO-C14-R1


title: HCT116-ZIC2KO-C3-R2


title: HCT116-ZIC2KO-C14-R2


title: HCT116-ZIC2-WT-R1

value: wild type


title: Sample group 35

city: Oxford

first: Sean

last: Wen

address: Headley Way

institute: University of Oxford

description: TARGET-seq

data_processing:
For processing of gDNA reads, sequencing reads were first aligned to the human reference genome using Burrows-Wheeler Aligner (BWA). Next, gDNA reads were retrieved based on gDNA-specific primer coordinates using custom perl script. Reads names were assigned and duplicate reads were marked using Picard.
For processing of mRNA reads, sequencing reads were first aligned to the human reference genome using STAR aligner. Next, cDNA reads were retrieved based on cDNA-specific primer coordinates using custom perl script. Reads names were assigned and duplicate reads were marked using Picard. Additionally, splicing intervals encoded as N CIGAR string were hardclipped using GATK-SplitNCigarReads.
Variant calling was performed using Mutect2 for insertion and deletion (INDEL) detection whereas Samtools mpileup was used for single-nucleotide substitution (SNV) detection.
Finally, Coverage at variant sites were computed using bedtools.
Assembly: GRCh38
Supplementary files format and content: Sparse matrix containing normalised umi counts for each cell

source_name: Bone marrow

extract_protocol: TARGET-seq (Rodriguez-Meira et al, 2019)

value: Bone marrow

value: Mononuclear cells

value: preleukemic


title: Sample group 32

description: TARGET-seq


title: HCT116-ZIC2-WT-R2


title: Sample group 40

description: TARGET-seq


title: Sample group 25

description: TARGET-seq

source_name: Peripheral blood

value: Peripheral blood


title: Sample group 19

description: TARGET-seq


title: w12_WD rep2

description: w12_WD rep2


title: Sample group 21

description: TARGET-seq


title: w12_CD+VC rep3

description: w12_CD+VC rep3


title: Sample group 16

description: TARGET-seq


title: Sample group 18

description: TARGET-seq


title: Sample group 15

description: TARGET-seq


title: Sample group 13

description: TARGET-seq

value: unassigned


title: Sample group 11

description: TARGET-seq


title: Sample group 4

description: TARGET-seq


title: Sample group 7

description: TARGET-seq


title: Background_50-75kDa_Rep2

city: Lausanne

first: Christina

last: Ernst

state: Vaud

address: EPFL SV GHI LVG, AI 3147 (Bâtiment AI), Station 19

department: School of Life Science

institute: EPFL

data_processing:
Sequencing data was demultiplexed by i7 index reads using bcl2fastq without any read trimming. Further demultiplexing by in-read 5’ barcodes and trimming of adapter sequences was performed using Flexbar v.3.5.0 (https://github.com/seqan/flexbar)20 in a two-step approach. In the first step, reads are demultiplexed by in-read barcodes allowing no mismatches, and UMIs are moved into the read header. Barcode sequences (see Supplementary Table 2) including the UMI designated by the wildcard character ’N’ are provided in fasta format, with the arguments “-b barcodes.fasta --barcode-trim-end LTAIL --barcode-error-rate 0 --umi-tags”. In the second step, any adapter contamination at the 3’ end of the reads is removed allowing an error rate of 0.1 with the following arguments “--adapter-seq 'AGATCGGAAGAGCACACGTCTGAACTCCAGTCACNNNNNNATCTCGTATGCCGTCTTCTGCTTG' --adapter-trim-end RIGHT --adapter-error-rate 0.1 --adapter-min-overlap 1”. In addition, potential T-stretches at the 5’ end that are the result of ribotailing during ligation are removed by trimming ‘T’ homopolymers of 1-2 nucleotide length (Supplementary Fig. 3) using “--htrim-left T --htrim-max-length 2 --htrim-min-length 1” and reads shorter than 18 nucleotides post trimming are discarded by “--min-read-length 18”.
Flexbar-trimmed reads were aligned against hg19 using STAR v.2.7.3a (https://github.com/alexdobin/STAR)21 with the following parameters, to keep only uniquely mapping reads, removing the penalty for opening deletions and insertions and fully extending the 5-prime end of reads to preserve the end of cDNA molecules: “--outFilterMultimapNmax 1 --scoreDelOpen 0 --scoreInsOpen 0 --alignEndsType Extend5pOfRead1”. To retain UMI in read header during STAR alignment, any space in header needs to be removed prior to mapping.
Aligned reads were deduplicated based on unique molecular identifiers using UMI-tools v.1.0.1 (https://github.com/CGATOxford/UMI-tools)22. The dedup command was used with the parameters “--extract-umi-method read_id --method unique --spliced-is-unique” to group reads with the same mapping position and identical UMI, while treating reads starting at the same position as unique if one is spliced and the other is not.
Enriched regions were identified using the peak calling algorithm CLIPper v. 2.0.0 (https://github.com/YeoLab/clipper)8,17 with default settings and a p-value cutoff of 0.01 “--poisson-cutoff 0.01”.
Assembly: hg19
Supplementary files format and content: bed format - Peaks obtained from CLIPper

growth_protocol: Adherent 293T cells (ATCC® CRL-1573™) were grown to ~80% confluency in Dulbecco’s Modified Eagle Medium (Gibco, #41966-029) supplemented with 10% FCS (Sigma-Aldrich, #F9665-500ML, Lot #19A124) and 1% Penicillin-Streptomycin-L-Glutamine (MED30-009-CI).

extract_protocol:
Protein-G beads (Thermo Fisher, 10004D) were washed twice in 1ml iCLIP Lysis buffer and resuspended in 100 µl per condition (100 µl of protein-G beads bind 20-30 µg of antibody - scale accordingly). Per IP, 1 µg of antibody against hnRNPc (Santa Cruz Biotechnology, sc-32308), RBM9 (Bethyl Laboratories, A300-864A), hnRNPA1 (4B10) (Santa Cruz Biotechnology, sc-32301), or hnRNPI (Santa Cruz Biotechnology, sc-56701) were added, and antibody-bead mixture was incubated at room temperature (RT) for 30-60 minutes on a rotating wheel. Antibody-bead mixture was washed twice in iCLIP lysis buffer to remove unbound antibody and RNase-treated cell lysates were added alongside cOmplete EDTA-free Protease Inhibitor Cocktail (Merck, #11836170001) and incubated for 2 hours at 4ºC on a rotating wheel. After IP, beads were washed twice in 200 µl High Salt Buffer (50 mM Tris-HCl pH 7.4, 1 M NaCl, 1 mM EDTA, 1% Igepal CA-630, 0.1% SDS, 0.5% sodium-deoxycholate), with the second wash at 4ºC for 3 minutes on a rotating wheel, followed by two washes in 200 µl PNK Wash Buffer (20 mM Tris-HCl, pH 7.4, 10 mM MgCl2, 0.2% Tween-20). Dephosphorylation of 3’ ends was performed in 20 µl of PNK reaction for 30 minutes at 37ºC (70 mM Tris-HCl, pH 6.5, 10 mM MgCl2, 1 mM DTT, 10U SUPERaseIN RNase Inhibitor (ThermoFisher, #AM2696), 5U T4 Polynucleotide Kinase (NEB, #M0201L). Beads were washed twice in PNK Wash Buffer and resuspended in 20 µl of ligation mix for overnight incubation at 16ºC and 1200 rpm (50 mM Tris-HCl, pH 7.8, 10 mM MgCl2, 1mM DTT, 10U SUPERaseIN RNase Inhibitor, 10U T4 RNA Ligase (NEB, #M0204), 1 µl of 1 µM L3 adapter and 20% PEG400 (Sigma-Aldrich, #91893)).
Following the first adapter ligation, beads were washed twice in 200 µl High Salt Buffer, twice in 200 µl PNK Wash buffer and then resuspended in 20 µl 1X LDS sample buffer (Thermo Fisher, #NP0008) containing 5% beta-mercaptoethanol (Sigma-Aldrich, #M6250). Samples were denatured for 1 minute at 70ºC and RNA-protein complexes were resolved on NuPAGE 4-12% Bis-Tris Gels (Thermo Fisher, #WG1402A) at 180V for 1 hour. Transfer was performed onto nitrocellulose (BioRad, #1620115) in 1X NuPAGE transfer buffer (Thermo Fisher, #NP00061) with 10% methanol at 30V for 2 hours at RT.   Nitrocellulose membranes were scanned on Odyssey® CLx Infrared Imager (LI-COR, 9141) with 169 µm resolution to visualise RNA localisation and then placed on filter paper soaked in PBS. Regions of interest were cut out from nitrocellulose membrane corresponding to ~20-100 kDa above the molecular weight of the RBP of interest due to the ligation of L3 adapter (~15.9 kDa) and associated RNA (with 70nt of RNA averaging ~20kDa). Nitrocellulose pieces were placed in LoBind Eppendorf tubes and 200 µl Proteinase K buffer (100mM Tris-HCl, pH 7.4, 50 mM LiCl, 1 mM EDTA, 0.2% LiDS) containing 200 µg Proteinase K (Thermo Fisher, #AM2546) were added and incubated at 50ºC for 45 minutes at 800rpm.  Meanwhile, 10 µl of Oligo(dT)25 Dynabeads™ (Thermo Fisher, #61005) per sample were washed in 1 ml of oligo(dT) Binding Buffer (20 mM Tris-HCl, pH 7.4, 1 M LiCl, 2 mM EDTA) and resuspended in 50 µl of oligo(dT) Binding Buffer per sample. Following Proteinase K treatment, supernatant was transferred to fresh tubes containing 50 µl of oligo(dT) beads and incubated for 10 minutes at RT on a rotating wheel. Following RNA capture, beads were washed twice in 125 µl oligo(dT) Wash Buffer (10 mM Tris-HCl, pH 7.4, 150 mM LiCl, 0.1 mM EDTA) and once in 20 µl 1X First-Strand Buffer (50 mM Tris-HCl, pH 8.3, 75 mM KCl, 3 mM MgCl2). Beads were resuspended in 10 µl of Reverse Transcription Mix (1X First-Strand Buffer, 0.5 mM dNTPs, 1 mM DTT, 6U SUPERase IN RNase Inhibitor, 20U SuperScript™ IV Reverse Transcriptase (Thermo Fisher, #18090050) and incubated for 15 minutes at 50ºC followed by 10 minutes at 80ºC heating up to 96ºC. Samples were vortexed for 30 seconds at 96ºC and then immediately placed on a magnet on ice. Supernatant containing adapter-ligated RNA was removed and efficiency of elution can be confirmed by dot-blotting on nitrocellulose membrane.   Solid-phase cDNA was washed once in 60 µl oligo(dT) Wash Buffer and once in 20 µl 1X T4 RNA Ligase Buffer (50 mM Tris-HCl, 10 mM MgCl2, 1mM DTT, pH 7.5). Beads were resuspended in 5 µl of 5’ Adapter mix (2 µl 10X T4 RNA Ligase Buffer, 2 µl of 10 µM L## oligo (see Supplementary Table 2), 1 µl 100% DMSO), incubated at 75ºC for 2 minutes then immediately placed on ice. 4 µl of Ligation Mix (5 mM ATP, 7U Terminal Deoxynucleotidyl Transferase (TdT) (2230B, Takara), 15 U T4 RNA Ligase High Concentration (M0437, NEB)) were added as well as 10 µl 50% PEG8000 and reaction was mixed by pipetting up- and down until beads are resuspended. Reaction was incubated at 37ºC for 30 minutes, then cooled down to room temperature. 30 U of T4 RNA Ligase were added, the reaction mixed by pipetting and incubated at RT overnight with occasional vortexing for 15 seconds at 2000 rpm every two minutes.  Following overnight incubation, ligation reaction was removed and beads washed in 100 µl oligo(dT) Wash Buffer and 20 µl 1X Phusion HF Buffer (Thermo Fisher, #F518L). Beads were resuspended in 25 µl cDNA amplification mix (1X Phusion HF PCR Master Mix (NEB, #M0531L) and 0.5uM P5 and P7 short primer mix (see Supplementary Table 2)) and amplification was performed with the following programme: 30 seconds at 98ºC, 7 cycles of 10 seconds at 98ºC, 30 seconds at 65ºC and 30 seconds at 72ºC followed by final extension at 72ºC for 3 minutes. Meanwhile, 2µl of oligo(dT) beads per sample were washed once in 1 ml oligo(dT) Binding buffer and resuspended in 5 µl per sample. After cDNA amplification, 5 µl of oligo(dT) beads were added and incubated at RT for 5 minutes on a rotating wheel to capture unwanted amplification by-products (see Fig. S3). Samples were placed on magnet and supernatant containing amplified cDNA was transferred to a fresh tube.   Size-selection of cDNA was performed using ProNEX® Size-Selective Purification System (Promega, #NG2002) with a ratio of 2.8X to enrich for cDNA inserts of at least 20 nucleotides in length (>80bp). Library yield was then estimated by amplifying 1 µl of purified cDNA via qPCR using the full length P5 and P7 index primers and 2-3 cycles were subtracted from the obtained Ct value for final library amplification. Following PCR amplification, libraries were size-selected again using the ProNEX® Size-Selective Purification System, with a ratio of 1.8X to select fragments larger than 165bp.
CLIP-seq

treatment_protocol: Cells were rinsed in ice-cold PBS and crosslinked on ice with 254 nm UV-C light at 0.3 J/cm2 in a CL-3000 Ultraviolet Crosslinker (UVPA849-95-0615-02). Cells were collected into PBS by scraping, counted and desired cell number was aliquoted and spun down. Cell pellets were resuspended in iCLIP Lysis buffer (50 mM Tris-HCl pH 7.4, 100 mM NaCl, 1% Igepal CA-630, 0.1% SDS, 0.5% sodium deoxycholate) using 50 µl per 50.000 cells. Lysates were incubated on ice for 5 minutes followed by sonication for 5-10 seconds at 0.5 seconds ON and 0.5 seconds OFF at 10% amplitude using a tip sonicator (Branson LPe 40:0.50:4T).

value: Human embryonic kidney

tag: clip antibody

value: hnRNP C1/2 (4F4) (SantaCruz, sc-32308)

tag: cell number

tag: rnase [u]


title: Background_50-75kDa_Rep1


title: Sample group 36

description: TARGET-seq


title: Background_75-100kDa_Rep2


title: Background_75-100kDa_Rep1


title: Background_100-150kDa_Rep2


title: Background_100-150kDa_Rep1


title: noUV_noPAGE_RBFOX2_Rep1

value: RBM9 (Bethyl Laboratories, #A300-864A)


title: noUV_noPAGE_RBFOX2_Rep2


title: Background_150-250kDa_Rep1


title: noUV_noPAGE_hnRNPc_Rep2


title: noUV_noPAGE_hnRNPI_Rep1

value: hnRNPI (SH54) (SantaCruz, sc-56701)


title: noUV_noPAGE_hnRNPA1_Rep2

value: hnRNPA1 (4B10) (SantaCruz, sc-32301)


title: noUV_noPAGE_hnRNPI_Rep2


title: noUV_noPAGE_hnRNPc_Rep1


title: noUV_hnRNPI_Rep2


title: noUV_RBFOX2_Rep2


title: noUV_RBFOX2_Rep1


title: noUV_noPAGE_hnRNPA1_Rep1


title: Sample group 14

description: TARGET-seq


title: noUV_hnRNPI_Rep1


title: noUV_hnRNPA1_Rep2


title: noUV_hnRNPc_Rep2


title: noUV_hnRNPA1_Rep1


title: RBFOX2_1k_Rep2


title: RBFOX2_10k_Rep2


title: RBFOX2_1k_Rep1


title: Background_150-250kDa_Rep2


title: hnRNPc_1k_Rep1


title: RBFOX2_10k_Rep1


title: hnRNPc_10k_Rep2