title: Batch1_Patient11600-15149_CD11_1

description:
CD11_1Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: PDX, adagrasib, rep 1

status: Public on Nov 01 2023

type: SRA

city: New York

first: ZHUXUAN

last: LI

state: NY

address: 417 E 68th St

department: Cancer Biology and Genetics

country: USA

institute: Memorial Sloan Kettering Cancer Center

library_source: transcriptomic

data_processing: PDXes and autochthonous KP LUAD tumors were FACS-isolated, RNA was extracted, and libraries were prepared for SMARTerSeq2, n = 3 mice/group. FASTQ files were processed using the standard DESeq2 pipeline (version 1.24.0). Reads were aligned to the GRCm38 mouse genome. Count matrices were generated was performed using DESeq2. Data were filtered to only include genes with 10 or more counts in 2 or more samples. The significance of differential expression between vehicle and treatment groups was quantified by one-way ANOVA.

molecule: polyA RNA

organism: Homo sapiens

source_name: PDX

extract_protocol:
To dissociate tumors into single-cell suspensions, primary tumors were finely chopped with scissors and incubated with digestion buffer containing collagenase IV (#17104019, ThermoFisher Scientific, 0.1 U/ml), dispase (#354235, Corning, 0.6 U/ml), and DNase I (#69182–3; Sigma Aldrich, 10 U/ml). For dissociating normal lung epithelial cells, digestion buffer was administrated intratracheally into the lung as described before (27). Following enzymatic dissociation, samples were washed with 2% heat-inactivated FBS in S-MEM (#11380037, Thermo Fisher Scientific), filtered through a 100 μm cell strainer (#431752, Corning), and centrifuged at 1500 rpm for 5 min at 4 °C. The supernatant was removed, and the pellet was resuspended in lysis buffer (#555899, BD Biosciences) to remove red blood cells. Cells were passed through a 40 μm strainer (#431750, Corning) and centrifuged at 1500 rpm for 5 min at room temperature. Cells were resuspended in 2% of heat-inactivated FBS in PBS. Cell suspensions were blocked for 5 min at 4 °C with rat anti-mouse CD16/CD32 (#553142, Mouse BD Fc Block, BD Biosciences) in FACS buffer, and incubated for 30 min with a mix of four APC-conjugated antibodies binding CD45 (#103112, Biolegend), CD31 (#102410, Biolegend), CD11b (#101212, Biolegend), F4/80 (#123116, BioLegend), CD19 (#115512, Biolegend), and TER-119 (#116212, Biolegend). 300 nM DAPI was added as a live-cell marker. Individual cancer cell suspensions were incubated for 30 min with hashtag oligonucleotide-conjugated antibodies in addition to FACS antibodies.
Sorted cell suspensions were prepared for scRNA-seq using the 3′ v3 10X Genomics Chromium platform according to manufacturer’s instructions. Briefly, sorted cells were washed once with PBS containing 1% bovine serum albumin (BSA) and resuspended in PBS containing 1% BSA to a final concentration of 700–1,300 cells per μl. The viability of cells in all experiments was above 80%, as confirmed with 0.2% (w/v) Trypan Blue staining (Countess II, Invitrogen). Cells were captured in droplets. Following reverse transcription and cell barcoding in droplets, emulsions were broken, and cDNA purified using Dynabeads MyOne SILANE followed by PCR amplification as per manufacturer’s instructions. Between 20,000 to 25,000 cells were targeted for each droplet formulation lane. Samples were multiplexed together in the lanes following the TotalSeq B cell hashing protocol. Final libraries were sequenced on Illumina NovaSeq S4 platform (R1 – 28 cycles, i7 – 8 cycles, R2 – 90 cycles).

tag: tissue

value: PDX

tag: cell type

tag: treatment

value: Adagrasib


title: PDX, vehicle, rep 1

value: vehicle


title: GBX2, day32, rep2

city: Aarhus

first: Mark

last: Denham

address: Høegh-Guldbergs Gade 10

department: Biomedicine

country: Denmark

institute: Aarhus University

description: column F, TPM_count_Table_4X_GBX_H9_TAB.txt

data_processing:
The reads were aligned to the Human genome build hg38 (Ensembl release 92) using HISAT2 aligner (v2.1.0). Transcript quantification was performed using FeatureCount (v1.6.4), and the read counts were normalized for effective gene length and sequencing depth to yield transcripts per kilobase million (TPM).
Assembly: hg38
Supplementary files format and content: count table
Supplementary files format and content: tab-delimited text file include TPM values for each Sample

source_name: hESCs

extract_protocol:
RNA was extracted using the Qiagen RNeasy mini kit and treated with DNase I according to the protocol
RNA-Seq libraries were prepared using the KAPA RNA HyperPrep kits (Roche) producing paired-end libraries. The input RNA was isolated from total purified RNA using poly(dA) selection.

value: hESCs

tag: cell line

value: caudally patterned neurons

tag: genotype

value: GBX2 knockout

value: 32 day differentiation


title: Batch1_Patient11600-15149_CD10_2

city: Oxford

first: Ahmed

middle: Ashour

last: Ahmed

state: Oxfordshire

address: John Radcliffe Hospital, Headley way

department: Weatherall Institute of Molecular Medicine

country: United Kingdom

institute: University of Oxford

description:
CD10_2Aligned.sortedByCoord.out.bam
counts_batch1.csv

library_source: transcriptomic single cell

data_processing:
Paired-end reads from Illumina NextSeq 500 were processed and mapped using STAR
The following filters were used to eliminate cells with (1) UMI counts < 10,000 and (2) gene counts < 200
Removed genes detected in < 3 cells
Assembly: UCSC hg19
Supplementary files format and content: Gene-cell matrix table containing transcript counts for every gene in every cell for each sample in batch1
Supplementary files format and content: Gene-cell matrix table containing transcript counts for every gene in every cell for each sample in batch2

source_name: Fallopian tube

growth_protocol: Fallopian tube WNT/β-catenin signaling reporter organoids were established from patient-derived cells, and expanded for 4-7 passages (each passage ~ 12 days). Endogenous WβA and non-WβA cells were isolated by FACS into RNA lysis buffer.

extract_protocol:
Isolated WβA or non-WβA cells were sorted into 96-well plates containing 4 μl lysis buffer supplemented with 0.1 μl RNase inhibitor (Clonetech), 1.9 μl 0.4% Triton X-100, 1 μl 10 μM 5‘-biotinylated oligo-dT30VN (IDT) and 1 μl 10 mM dNTP (Thermo Scientific). Cells were sorted at one cell per well, with bulk controls (10 cells) and empty well controls (0 cells) included for each plate. Plates were snap frozen on dry ice and stored at - 80 degrees C for less than 4 weeks.
Single cell cDNA synthesis and library generation were performed according to the SMART-seq2 protocol (Picelli et al., 2014), as previously described (Hu et al., 2020). Briefly, cells were lysed by removing plates from  -80 0C and heating at 72 0C for 3 min. Plates were then placed at 4 0C before adding the reverse transcription mix containing 5’-biotinylated TSO (Qiagen). PCR products were cleaned up using 0.8:1 Ampure XP beads (Beckman Coulter) with Biomek FxP Laboratory Automation Workstation (Biomek). Quality of single-cell cDNA was tested using TapeStation, as well as by single cell qPCR for GAPDH or ACTB using the QuantiNova SYBR Green PCR Kit (Qiagen). cDNA concentration was measured using Quant-iTTM PicoGreenTM dsDNA Assay Kit (Invitrogen) on the CLARIOstar Plate Reader (BMG Labtech). Wells with CT values of GAPDH or ACTB below 20 were selected as wells with good quality cDNA. Libraries from single cell cDNA were generated using miniaturized Nextera XT (Illumina) protocol (Mora-Castilla et al., 2016) with Mosquito HTS (TTP LabTech), in 384-well Endure plate (Life Technology). Library sequencing was performed by Novogene.

value: Fallopian tube


title: V12, mechanical ventilation_12hr_rep3

type: RNA

city: Nishinomiya

first: Yutaka

last: Hirata

state: Hyogo

address: 1-1 Mukogawa-cho

department: Integrative Physiology

country: Japan

institute: Hyogo Medical University

description: Gene expression data from diaphragm

hyb_protocol: GeneChip™ arrays (Rat ClariomTM S) were hybridized with biotin-labeled cDNA samples at 45°C for 16 hours (60 rpm) using a GeneChip™ Hybridization Oven 645.

scan_protocol: The arrays were washed and stained with a GeneChip™ Fluidics Station 450 and scanned on an Affymetrix Gene Chip Scanner 3000 7G using Command Console Software.

data_processing: The signal values of the GeneChip™ array were normalized based on the total intensity of the array using the SST-RMA algorithm implemented in Transcriptome Analysis Console (TAC) v4.0 software (Thermo Fisher Scientific, MA, USA).

label: biotin

molecule: total RNA

organism: Rattus norvegicus

source_name: Rats with tracheostomy, mechanically ventilated for 12 hours

label_protocol: From 250 ng of total RNA in each group, fragmented and biotin-labeled complementary deoxyribonucleic acid (cDNA) samples were synthesized using a GeneChip™ WT PLUS Reagent Kit according to the protocol provided with the kit.

extract_protocol: Total RNA was isolated from the minced muscle tissue using a NucleoSpin RNA Kit (Takara, Japan) according to the manufacturer’s instructions.

value: diaphragm

tag: strain

value: Wistar

tag: age

value: 14-16 weeks old

tag: gender

value: male


title: Normal Control 3

city: Xi'an

first: Xi

last: Wang

address: No.76 Yanta west road

country: China

institute: Xi'an Jiaotong University

data_processing:
Raw reads were subjected to an in-house program, ACGT101-miR (LC Sciences, Houston, Texas, USA) to remove adapter dimers, junk, low complexity, common RNA families (rRNA, tRNA, snRNA, snoRNA) and repeats.
Subsequently, unique sequences with length in 18~26 nucleotide were mapped to specific species precursors in miRBase 22.0 by BLAST search to identify known miRNAs and novel 3p- and 5p- derived miRNAs.
Length variation at both 3’ and 5’ ends and one mismatch inside of the sequence were allowed in the alignment. The unique sequences mapping to specific species mature miRNAs in hairpin arms were identified as known miRNAs.
The unique sequences mapping to the other arm of known specific species precursor hairpin opposite to the annotated mature miRNA-containing arm were considered to be novel 5p- or 3pderived miRNA candidates.
The remaining sequences were mapped to other selected species precursors (with the exclusion of specific species) in miRBase 22.0 by BLAST search, and the mapped pre-miRNAs were further BLASTed against the specific species genomes to determine their genomic locations.
Assembly: miRBase 22.0
Supplementary files format and content: tab-delimited text files for each Sample

source_name: chondrocyte

extract_protocol:
The total RNA was extracted using the Total RNA Purification Kit (LC Sciences, Houston, USA), according to the manufacturer’s protocol. The total RNA quantity and purity were analysis of Bioanalyzer 2100 and RNA 6000 Nano LabChip Kit (Agilent, CA, USA) with RIN number >7.0.
Approximately 1 ug of total RNA were used to prepare small RNA library according to protocol of TruSeq Small RNA Sample Prep Kits (Illumina, San Diego, USA). And then we performed the single-end sequencing (1x50bp) on an Illumina Hiseq2500 at the LC-BIO (Hangzhou, China) following the vendor’s recommended protocol.

tag: disease

value: Normal

value: chondrocyte


title: tRNA Neo1.0 linear, rep 2

city: Chicago

first: Tao

last: Pan

state: Illinois

address: 929 E. 57th Street

department: Biochemistry and Molecular Biology

institute: University of Chicago

data_processing:
Raw sequences reads were demultiplexed using Je to detect encoded barcode reads. Following this, reads were aligned to curated Sacchromyces tRNA reference genome.
The aligned reads were then used to determine RNA sequence abundance using custom python script. RNA modifications were detected based on aligned reads using samtools sort (http://www.htslib.org/,Li et al., 2009) feature sort the reads in a bam file format. Then IGVtools count (https://software.broadinstitute.org/software/igv/igvtools) count feature was utilized to output a wig files using the following parameters: -z 5 -w 1 -e 250 —bases. The resulting wig files were processed using a custom python script to identify nucleotide mutations as well as coverage of aligned reads.
Supplementary files format and content: tsv files with gene name and abundance per sample
Supplementary files format and content: tsv files with mutation, deletion, coverage for each gene position per sample

organism: Saccharomyces cerevisiae

growth_protocol: All yeast strains were derivatives of S288C and grown at 30°C unless otherwise specified. A full list of strains used may be found in Table S1. Yeast cells were grown in either YEP/YPD media (10 g/L yeast extract, 20 g/L peptone and with or without 0.64 g/L tryptophan) or Synthetic Complete (SC) media lacking the indicated amino acids, both supplemented with 2% glucose if not stated otherwise. Standard solid media contained 2% agar and cultures were incubated at 30°C. Liquid cultures were cultivated according to standard procedures in glass tubes on a TC-7 roller drum (New Brunswick).

extract_protocol:
Total RNA was extracted from cells using Tri-zol extraction.
MSR-seq (PMID 35513407)

treatment_protocol: Yeast transformation was undertaken according to a modified protocol described by Daniel Gietz and Woods (2002). Briefly, yeast strains were inoculated into 10 mL of liquid media and incubated with rotation at 30°C overnight. Overnight cultures were then re-inoculated to OD600 = 0.1 and incubated with rotation to a target OD600 of between 0.5 and 1. Cells were then centrifuged at 2,103 g for 5 minutes, washed with 10 mL sterile ddH2O and centrifuged again. Cell pellets were washed with 10 mL of 0.1 M LiOAc, centrifuged at 2,103 g for 5 minutes. 50 μL of cells were mixed with 5 to 10 μL of transforming DNA, 36 μL of 1M LiOAc, 19 μL ddH2O, 25 μL 10 mg/mL herring sperm carrier DNA and 240 μL of 44% PEG 3350. Transformations were incubated at 30°C for 30 minutes before adding 36 μL of DMSO and heat-shock at 42°C for 15 minutes. Cells were pelleted and incubated in 400 μL of 5 mM CaCl2 at room temperature for 10 minutes and plated onto selective media.

value: yeast cells

value: tRNA neo1.0 linear


title: Normal Control 1


title: Batch1_Patient11600-15149_CD09_1

description:
CD09_1Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: Batch1_Patient11600-15149_CD09_2

description:
CD09_2Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: Batch1_Patient11600-15149_CE02_1

description:
CE02_1Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: KBD3

value: Kashin-Beck disease


title: PDX, vehicle, rep 3

value: PDX


title: Batch1_Patient11600-15149_CD12_1

description:
CD12_1Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: Batch1_Patient11600-15149_CE01_2

description:
CE01_2Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: KBD2


title: Batch1_Patient11600-15149_CD08_1

description:
CD08_1Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: Batch1_Patient11600-15149_CD12_2

description:
CD12_2Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: KBD1


title: PDX, vehicle, rep 2


title: Batch1_Patient11600-15149_CD08_2

description:
CD08_2Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: Batch1_Patient11600-15149_CD07_2

description:
CD07_2Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: Batch1_Patient11600-15149_CD07_1

description:
CD07_1Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: Batch1_Patient11600-15149_CD11_2

description:
CD11_2Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: Batch1_Patient11600-15149_CD06_2

description:
CD06_2Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: XNES1_CR110_TRIM28i_H3K9me3_S13

city: Lund

first: Johan

last: Jakobsson

address: Sölvegatan 17

department: Wallenberg Neuroscience Center

country: Sweden

institute: Lund University

library_source: genomic

data_processing:
RNAseq Unique mapping (intron 32 counts): STAR aligner (--outFilterMultimapNmax 1, --outFilterMismatchNoverLmax 0.03). Mapping (gene and exon counts): STAR aligner, default parameters.
RNAseq Genes were quantified using featureCounts (Subread version 1.6.3), forcing matching strandness of the reads to the features being quantified (-s 2). Exons were quantified using featureCounts (Subread version 1.6.3), forcing matching strandness of the reads to the features being quantified (-s 2), attribute type set to exons (-g exon_id) (-f -O).
CUT&RUN Reads were aligned to the human and yeast genomes (hg38 and R64-1-1 respectively) using bowtie2 (–local –very-sensitive- local –no-mixed –no-discordant –phred33 -I 10 -X 700)
BAM files were indexed using samtools (version 1.12) and converted to bigwig files using deeptools bamCoverage (version 2.5.4) (--normalizeUsingRPKM)
Assembly: GRCh38
Supplementary files format and content: Bigwigs for genome browser visualization
Supplementary files format and content: Bulk RNAseq read count matrix for genes, exons and TAF1 intron 32.
Library strategy: CUT&RUN

molecule: genomic DNA

source_name: NPC

extract_protocol:
Total RNA was isolated using the RNeasy Mini Kit (Quiagen) with on column DNAse treatment following the manufacturer’s instructions.  RNA sequencing was performed using four biological replicates unless stated differently. CUT&RUN analysis was done following the protocol described in Skene&Henikoff  2018 (Skene & Henikoff, 2017). 300.000 cells were used to do the analysis.
Libraries for RNA sequencing were generated using Illumina TruSeq Stranded mRNA library prep kit (poly-A selection). For CUT&RUN analysis Illumina sequencing libraries were prepared using the Hyperprep kit (KAPA) with unique dual indexed adapters (KAPA).
The generated libraries were optimized for long fragments, and sequenced on a Novaseq6000 (paired end, ~ 150 bp) yielding an average of 46M reads. Cut&RUN libraries were sequenced on a Nextseq500 instrument (paired end, 2x75bp).

tag: individual

tag: dx

value: XDP

tag: cell type_at_ko/kd

value: NPC

tag: condition

value: CRISPRi

tag: target gene

tag: batch

value: batchG

tag: antibody


title: Batch1_Patient11600-15149_CD06_1

description:
CD06_1Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: XNES1_CR110_ZNF91i_H3K9me3_S15


title: XNES1_CR110_LacZ_IgG_S10

value: Control

value: LacZ

value: IgG


title: Batch1_Patient11600-15149_CD10_1

description:
CD10_1Aligned.sortedByCoord.out.bam
counts_batch1.csv

value: Fallopian tube


title: Normal Control 2


title: Batch1_Patient11600-15149_CD05_2

description:
CD05_2Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: XNES1_CR110_LacZ_H3K9me3_S11


title: Batch1_Patient11600-15149_CD05_1

description:
CD05_1Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: CNES1_CR110_ZNF91i_H3K9me3_S14


title: CNES1_CR110_TRIM28i_H3K9me3_S12


title: Batch1_Patient11600-15149_CD04_2

description:
CD04_2Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: CNES1_CR110_LacZ_H3K9me3_S9


title: Batch1_Patient11600-15149_CD04_1

description:
CD04_1Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: Batch1_Patient11600-15149_CD03_2

description:
CD03_2Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: XNES1_ZNF91_KD_A_iPS_S13

description:
gene_count_matrix_2_xdp.csv
exon_count_matrix_2_xdp.csv
intron32_count_matrix_2_xdp.csv

data_processing:
RNAseq Unique mapping (intron 32 counts): STAR aligner (--outFilterMultimapNmax 1, --outFilterMismatchNoverLmax 0.03). Mapping (gene and exon counts): STAR aligner, default parameters.
RNAseq Genes were quantified using featureCounts (Subread version 1.6.3), forcing matching strandness of the reads to the features being quantified (-s 2). Exons were quantified using featureCounts (Subread version 1.6.3), forcing matching strandness of the reads to the features being quantified (-s 2), attribute type set to exons (-g exon_id) (-f -O).
CUT&RUN Reads were aligned to the human and yeast genomes (hg38 and R64-1-1 respectively) using bowtie2 (–local –very-sensitive- local –no-mixed –no-discordant –phred33 -I 10 -X 700)
BAM files were indexed using samtools (version 1.12) and converted to bigwig files using deeptools bamCoverage (version 2.5.4) (--normalizeUsingRPKM)
Assembly: GRCh38
Supplementary files format and content: Bigwigs for genome browser visualization
Supplementary files format and content: Bulk RNAseq read count matrix for genes, exons and TAF1 intron 32.

source_name: iPSC

value: iPSC

value: batchF


title: Batch1_Patient11600-15149_CD03_1

description:
CD03_1Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: XNES1_ZNF91_KD_A_iPS_S10


title: Batch1_Patient11600-15149_CD02_2

description:
CD02_2Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: XNES1_LZ_D_iPS_S17


title: Batch1_Patient11600-15149_CD02_1

description:
CD02_1Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: XNES1_ZNF91_KD_A_iPS_S12


title: XNES1_ZNF91_KD_A_iPS_S11


title: Batch1_Patient11600-15149_CD01_2

description:
CD01_2Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: CNES1_CR110_LacZ_IgG_S8


title: Batch1_Patient11600-15149_CD01_1

description:
CD01_1Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: XNES1_LZ_C_iPS_S16


title: Batch1_Patient11600-15149_CC12_2

description:
CC12_2Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: Batch1_Patient11600-15149_CC12_1

description:
CC12_1Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: XNES1_LZ_B_iPS_S15


title: XNES1_ZNF91KD_in_iPS_D_S16

value: batchE


title: Batch1_Patient11600-15149_CC11_2

description:
CC11_2Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: XNES1_ZNF91KD_in_iPS_C_S15


title: Batch1_Patient11600-15149_CC10_2

description:
CC10_2Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: Batch1_Patient11600-15149_CC11_1

description:
CC11_1Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: XNES1_ZNF91KD_in_iPS_B_S14


title: XNES1_ZNF91KD_in_iPS_A_S13


title: Batch1_Patient11600-15149_CC10_1

description:
CC10_1Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: Batch1_Patient11600-15149_CC09_2

description:
CC09_2Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: XNES1_ZF91KD_LZD_iPS_S37

value: batchD


title: Batch1_Patient11600-15149_CC09_1

description:
CC09_1Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: Batch1_Patient11600-15149_CC08_2

description:
CC08_2Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: XNES1_ZF91KD_LZC_iPS_S32


title: Batch1_Patient11600-15149_CC08_1

description:
CC08_1Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: XNES1_ZF91KD_LZB_iPS_S27


title: XNES1_ZF91KD_G1C_iPS_S12


title: XNES1_ZF91KD_LZA_iPS_S22


title: Batch1_Patient11600-15149_CC07_2

description:
CC07_2Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: RYGB ileum FDG increased_replicate3

city: Seoul

first: Chan Woo

last: Kang

state: Seoul

address: 50 Yonsei-ro Seodaemun-gu

country: South Korea

institute: Yonsei University

data_processing:
Raw images were generated using the HiSeq Control software v2.2.38, base calling using an integrated primary analysis software, Real time Analysisv1.18.61.0, and converted into FASTQ files using the Illumina package bcl2fastq v1.8.4.
The alignment of reads against the human reference genome hg19 was performed using the STAR 2.6.0a software.
Reads Per Kilobase of exon per Megabase of library size (RPKM) were calculated using a protocol from Chepelev et al., Nucleic Acids Research, 2009. In short, exons from all isoforms of a gene were merged to create one meta-transcript. The number of reads falling in the exons of this meta-transcript were counted and normalized by the size of the meta-transcript and by the size of the library.
Gene expression quantification using Cufflinks 2.2.1.
The differential expression analysis was conducted by Cuffdiff and DESeq2 (version: 1.24.0).
Genome_build: Genome_build: hg19
Supplementary_files_format_and_content: tab-delimited text files include FPKM values for each Sample

source_name: RYGB ileum FDG increased

extract_protocol:
ileum were removed, flash frozen on dry ice, and RNA was harvested using Qiagen RNA extraction kit. ITotal RNA sequencing libraries were generated using Illumina TruSeq Stranded Total RNA Sample prep kit with Ribo-zero Human/Mouse/Rat,
RNA libraries were prepared for sequencing using standard Illumina protocols

treatment_protocol: 1month after Roux-en-Y operation

value: Otsuka Long-Evans Tokushima fatty

value: Roux-en-Y operation

value: small intestine (ileum)


title: XNES1_ZF91KD_G1D_iPS_S17

description:
gene_count_matrix_2_xdp.csv
exon_count_matrix_2_xdp.csv
intron32_count_matrix_2_xdp.csv


title: RYGB ileum FDG increased_replicate2


title: XNES1_DNMT1_ZF91_LZ__D_S39


title: Batch1_Patient11600-15149_CC06_2

description:
CC06_2Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: XNES1_ZF91KD_G1A_iPS_S2


title: Batch1_Patient11600-15149_CC07_1

description:
CC07_1Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: XNES1_DNMT1_ZF91__LZ__C_S34


title: XNES1_ZF91KD_G1B_iPS_S7


title: XNES1_DNMT1_ZF91_LZ__B_S29


title: XNES1_DNMT1_ZF91_LZ__A_S24


title: RYGB ileum FDG increased_replicate1


title: Batch1_Patient11600-15149_CC06_1

description:
CC06_1Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: XNES1_DNMT1_ZF91_KD__D_S19


title: RYGB_ileum_FDG decreased_replicate2

source_name: RYGB_ileum_FDG decreased


title: XNES1_LZ_A_iPS_S14


title: Batch1_Patient11600-15149_CC05_2

description:
CC05_2Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: Batch1_Patient11600-15149_CC05_1

description:
CC05_1Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: XNES1_DNMT1_ZF91_KD__C_S14


title: RYGB_ileum_FDG decreased_replicate1


title: XNES1_DNMT1_ZF91_KD__B_S9


title: XNES1_DNMT1_ZF91_KD__A_S4


title: Batch1_Patient11600-15149_CC04_2

description:
CC04_2Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: XNES1_DNMT1_LZ__B_S28


title: Sham_ileum_replicate3

value: sham operation


title: Batch1_Patient11600-15149_CC04_1

description:
CC04_1Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: XNES1_DNMT1_LZ__A_S23


title: Sham_ileum_replicate2


title: Batch1_Patient11600-15149_CC03_2

description:
CC03_2Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: TLR4.6

city: Shanghai

first: Yunjie

last: Shi

address: No.800 Xiangyin Road

institute: Naval Medical University

data_processing:
Using FLASH v1.2.7 software, the reads of each sample are spliced by overlap, and the spliced sequence is the raw Tags.Using Trimmomatic v0.33 software, the Raw Tags obtained by splicing were filtered to obtain high-quality Tags data (Clean Tags)
Using UCLUST in QIIME(version 1.8.0) software to cluster Tags at the similarity level in ninety-seven percent, OTU was obtained, and the OTU was taxonomically annotated based on Silva (bacteria) and UNITE (fungi) taxonomic databases.
Comparing the representative sequence of the OTUs with a microbial reference database to obtain species classification information corresponding to each OTU, and counting the composition of each sample community at each level,
The Alpha diversity index of samples was evaluated using the Mothur (version v.1.30) software. In general, the index of diversity was compared among samples, and the number of sequences contained in samples was standardized for analysis.
The PICRUSt software was used to deduce the functional gene composition in samples by comparing the species composition information obtained from 16S sequencing data, so as to analyze the functional differences between different samples or subgroups.
Assembly: mm10
Library strategy: 16S rRNA sequencing

organism: feces metagenome

source_name: fecal samples of mice

extract_protocol:
Extraction of total genomic DNA through the DNA Extraction Kit (Tiangen Biotechnology Company, Beijing, China) was conducted following the instructions of the kit.
DNA libraries for 16s-rDNA sequencing were prepared using SMARTER DNA-Seq Library Prep Kit following manufacturer's protocols.

value: fecal samples of mice

tag: mouse genotype

value: TLR4 knockout

tag: mouse treatment

value: Dextran Sulfate Sodium Salt (DSS)-induced colitis


title: XNES1_DNMT1_KO__C_S13

value: XDP

value: NPC

tag: cell type

value: CRISPR-cut


title: SupT1, Latent-infected, rep2

city: Ithaca

first: Gabrielle

last: Lê-Bury

address: 930 campus road

institute: Cornell University

data_processing:
Raw RNA-seq reads were first analyzed with FastQC for quality control and the processed through Trim Galore for removal of low quality reads and the Illumina sequencing adapters
The trimmed, high-quality reads were aligned to the Ensembl human reference genome (GRCh38 v86) using the STAR aligner software
Raw reads count for each gene were obtained using -quantmode of GeneCounts
Assembly: GRCh38
Supplementary files format and content: tab-delimited text files include gene counts

growth_protocol: SupT1 cells were cultured in RPMI media supplemented with 10% fetal bovine serum (FBS) and 1% each of penicillin, streptomycin and glutamine

extract_protocol:
Total RNA was extracted from flow-sorted cells using TRIzol reagent (Invitrogen) according to manufacturer’s protocol, followed by treatment with DNase (Turbo DNAse-free kit, Invitrogen)Ribosomal RNA (rRNA) was removed from the total RNA (50ng) using NEBNext rRNA Depletion kit v2 (Human/Mouse/Rat, New England Biolab), according to manufacturer’s instructions.
The rRNA-depleted samples were used for stranded library preparation using NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England Biolabs).

treatment_protocol: SupT1 cells were infected with HIVGKO virus at an multiplicity of infection of 0.1 by spinoculation (3000rpm, 30min, 30C). Virus-infected or uninfected cells were cultured for 4 days in a 37C incubator with 6% CO2. Uninfected or HIV-1 active-infected (mKO2+GFP+) and latent- infected (mKO2+GFP-) SupT1 cells were flow sorted using a Biorad S3 cell sorter

value: T lymphoblast

value: WT

tag: treatment

value: HIV GKO infection

tag: time

value: 4 days post infection


title: TLR4.5

tag: tissue


title: XNES1_DNMT1_KO__D_S18


title: Sham_ileum_replicate1

tag: strain


title: RYGB_ileum_FDG decreased_replicate3


title: SupT1, Latent-infected, rep1

tag: cell line

tag: genotype


title: XNES1_DNMT1_KO__B_S8


title: TLR4.3


title: TLR4.4


title: Batch1_Patient11600-15149_CC01_2

description:
CC01_2Aligned.sortedByCoord.out.bam
counts_batch1.csv

value: Fallopian tube


title: Batch1_Patient11600-15149_CC02_2

description:
CC02_2Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: Batch1_Patient11600-15149_CC02_1

description:
CC02_1Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: SupT1, Active-infected, rep2


title: Batch1_Patient11600-15149_CC03_1

description:
CC03_1Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: CNES1_DNMT1_ZF91_LZ__D_S40

value: Control


title: TLR4.2


title: XNES1_DNMT1_KO__A_S3

value: batchD


title: TLR4.1


title: XNES1_DNMT1_LZ__C_S33


title: Batch1_Patient11600-15149_CC01_1

description:
CC01_1Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: SupT1, Active-infected, rep1


title: CNES1_DNMT1_ZF91__LZ__C_S35


title: SupT1, Uninfected, rep2


title: WT. 6


title: CNES1_DNMT1_ZF91_LZ__B_S30


title: Batch1_Patient11600-15149_CB12_2

description:
CB12_2Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: SupT1, Uninfected, rep1


title: WT. 5


title: CNES1_DNMT1_ZF91_LZ__A_S25


title: Batch1_Patient11600-15149_CB12_1

description:
CB12_1Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: WT. 4


title: CNES1_DNMT1_ZF91_KD__C_S15

value: CRISPRi


title: Batch1_Patient11600-15149_CB11_2

description:
CB11_2Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: WT. 3


title: HDMVEC, control(S)

city: Los Angeles

first: Gerard

last: Wong

state: CA

address: 410 Westwood Plaza

department: Bioengineering

institute: University of California Los Angles

data_processing:
Sequence reads are quantitated using Whippet (version 1.6.1) with the GRCh38.p13 RefSeq genome annotations.
Differential expression analysis and PCA are performed with DESeq2 (version 1.34.0).
KEGG pathway analysis is performed with significant differentially expressed genes (adjusted p-value < 0.05) using clusterProfiler (version 4.2.2).
Assembly: GRCh38.p13 RefSeq genome
Supplementary files format and content: Normalized abundance measurements output from DESeq

source_name: HDMVEC

growth_protocol: HDMVECs are maintained in Endothelial Cell Growth Medium MV 2 (PromoCell) in humidified atmosphere with 5% CO2 at 37C

extract_protocol:
Total cellular RNA is extracted using the PureLink RNA Mini Kit.
Libraries are constructed using TruSeq Stranded mRNA Library PrepKits (Illumina, San Diego, CA)

treatment_protocol: Cells are seed in 24 well plate at a density of 2.50 × 10^5 cells/well a day before the treatment. Cells are stimulated for 24 hrs with 2.5 µg/ml Poly(I:C) alone or complexed with xenoAMPs at 2x isoelectric ratio.

value: Primary human dermal microvascular endothelial cells (HDMVECs)

value: control(S)

value: 24 hours


title: HDMVEC, xenoAMP(S)

value: xenoAMP(S)


title: HDMVEC, control(S)-dsRNA

value: control(S)-dsRNA complex


title: WT. 2

value: fecal samples of mice

value: Dextran Sulfate Sodium Salt (DSS)-induced colitis


title: CNES1_DNMT1_ZF91_KD__A_S5

description:
gene_count_matrix_2_xdp.csv
exon_count_matrix_2_xdp.csv
intron32_count_matrix_2_xdp.csv

value: NPC


title: CNES1_DNMT1_ZF91_KD__B_S10


title: HDMVEC, untreated

value: untreated


title: Batch1_Patient11600-15149_CB10_1

description:
CB10_1Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: Batch1_Patient11600-15149_CB11_1

description:
CB11_1Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: CNES1_DNMT1_LZ__D_S36


title: HDMVEC, xenoAMP(S)-dsRNA

value: xenoAMP(S)-dsRNA complex


title: Batch1_Patient11600-15149_CB09_2

description:
CB09_2Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: Input_TG_thymocytes

city: Tianjin

first: Wei

last: Sun

address: No.94 Weijin Road

department: School of Medicine

institute: Nankai University

data_processing:
BGISEQ500 platform were used for sequencing and basecalling. Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence in this platform.
RNA-seq reads were mapped to mm10 reference genome using HISAT with recommanded parameters.
For RNA-seq, gene expression values were calculated with DEseq2.
ChIP-seq reads were aligned to the mm9 genome using Bowtie2 with recommanded parameters.
ChIP peaks were called by MACS2 with input, p<0.01 was set as the threshold.
Genome_build: mm10 for RNA-seq, mm9 for ChIP-seq
Supplementary_files_format_and_content: FPKM and counts matrix file for RNA-seq, peak file for ChIP-seq

organism: Mus musculus

source_name: thymocytes

growth_protocol: primary cells isolated from mouse fed in SPF condition

extract_protocol:
total RNA was extracted for RNA-seq, ChIP DNA was obtained with a SimpleChIP® Enzymatic Chromatin IP Kit (Cell Signaling Technology, Danvers, MA, USA) following the manufacturer’s instructions.
RNA libraries and ChIP DNA libraries were constructed for sequencing using standard protocols by BGI-tech (BGI, Shenzhen, Guangdong, China).

value: total primary thymocytes

value: hCD2-LMO2 knock-in


title: WT. 1

value: WT


title: CNES1_DNMT1_LZ__C_S31


title: TG_spleen_CD8_LMO2

source_name: splenic T cells

value: sorted primary splenic CD8+ T cells


title: Batch1_Patient11600-15149_CB09_1

description:
CB09_1Aligned.sortedByCoord.out.bam
counts_batch1.csv

value: Fallopian tube


title: CNES1_DNMT1_LZ__A_S21

value: Control

value: batchD


title: TG_spleen_CD4_LMO2

value: sorted primary splenic CD4+ T cells


title: U-3

city: Birmingham

first: Narendra

last: Wajapeyee

state: AL

address: 720 20th Street South, Kaul 540A

department: Department of Biochemistry and Molecular Genetics

institute: University of Alabama at Birmingham

data_processing:
The reads were first mapped to the latest UCSC transcript set using Bowtie2 version 2.1.0 and the gene expression level was estimated using RSEM v1.2.15.
Genome_build: hg38
Supplementary_files_format_and_content: tab-delimited text files include raw count values for each Sample

source_name: SK-MEL-147  cell lines

growth_protocol: SK-MEL-147 cells were grown in DMEM medium with 10% Fetal bovine serum and Penicilline and Streptomycin at  37 C under 5% CO2 condition.

extract_protocol:
Total RNA was extracted using Trizol reagent (Invitrogen) as per the instruction from the manufacture and then purified using RNAeasy mini columns (Qiagen) as per the manufactures instructions.
RNA libraries were prepared for sequencing using standard Illumina protocols

treatment_protocol: SK-MEL-147 cells used for preparing the total RNA and to perfrom gene expression array.

value: UNC0642 treatment


title: Batch1_Patient11600-15149_CB08_2

description:
CB08_2Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: U-2


title: CNES1_DNMT1_KO__C_S11

value: CRISPR-cut


title: Batch1_Patient11600-15149_CB10_2

description:
CB10_2Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: Batch1_Patient11600-15149_CB08_1

description:
CB08_1Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: U-1


title: WT_DN3_LMO2

value: sorted primary CD25+ DN thymocytes

value: wild type


title: CNES1_DNMT1_KO__B_S6


title: J-2

value: JQ-1 treatment


title: TG_DN3_LMO2


title: TG_S_CD8_3


title: Batch1_Patient11600-15149_CB06_2

description:
CB06_2Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: Batch1_Patient11600-15149_CB07_1

description:
CB07_1Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: HDMVEC, dsRNA

value: Primary human dermal microvascular endothelial cells (HDMVECs)

value: dsRNA

value: 24 hours


title: Batch1_Patient11600-15149_CB06_1

description:
CB06_1Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: CNES1_DNMT1_KO__D_S16

value: NPC


title: CNES1_1DNMT1_KO__A_S1


title: XNES3_ZF91KD_LZ_D_S34

value: XDP

value: batchC


title: TG_S_CD8_2

value: hCD2-LMO2 knock-in


title: XNES3_ZF91KD_LZ_C_S33


title: TG_S_CD8_1

value: sorted primary splenic CD8+ T cells


title: M-3

value: MS023 treatment


title: J-1


title: Batch1_Patient11600-15149_CB07_2

description:
CB07_2Aligned.sortedByCoord.out.bam
counts_batch1.csv

value: Fallopian tube


title: XNES3_ZF91KD_LZ_B_S32

value: Control


title: Batch1_Patient11600-15149_CB05_2

description:
CB05_2Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: J-3


title: M-2


title: TG_S_CD4_3

value: sorted primary splenic CD4+ T cells


title: TG_DP_LMO2

value: sorted primary DP thymocytes


title: XNES3_ZF91KD_LZ_A_S31


title: Batch1_Patient11600-15149_CB04_2

description:
CB04_2Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: Batch1_Patient11600-15149_CB05_1

description:
CB05_1Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: TG_S_CD4_2


title: M-1


title: TG_S_CD4_1


title: XNES3_ZF91KD_G1D_S30

value: CRISPRi


title: GK-3

value: GSK-484 treatment


title: Batch1_Patient11600-15149_CB04_1

description:
CB04_1Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: TG_CD8SP_3

value: sorted CD8SP primary thymocytes


title: Batch1_Patient11600-15149_CB03_2

description:
CB03_2Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: G-3

value: GSK-591 treatment


title: GK-2


title: TG_CD8SP_2


title: XNES3_ZF91KD_G1A_S27

description:
gene_count_matrix_2_xdp.csv
exon_count_matrix_2_xdp.csv
intron32_count_matrix_2_xdp.csv


title: Batch1_Patient11600-15149_CB02_2

description:
CB02_2Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: XNES3_ZF91KD_G1B_S28


title: GK-1


title: Batch1_Patient11600-15149_CB03_1

description:
CB03_1Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: CNES2_ZF91KD_LZ_D_S26


title: CNES2_ZF91KD_LZ_C_S25


title: TG_CD8SP_1


title: G-2


title: G-1


title: WT_CD8SP_2

value: wild type


title: Batch1_Patient11600-15149_CB01_2

description:
CB01_2Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: CNES2_ZF91KD_LZ_B_S24


title: TP-3

value: TP-472 treatment


title: WT_CD8SP_1


title: Batch1_Patient11600-15149_CB01_1

description:
CB01_1Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: TP-2


title: XNES3_ZF91KD_G1C_S29


title: CNES2_ZF91KD_LZ_A_S23


title: Batch1_Patient11600-15149_CA12_2

description:
CA12_2Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: CNES2_ZF91KD_G1D_S22


title: TG_CD4SP_2

value: sorted CD4SP primary thymocytes


title: CNES2_ZF91KD_G1C_S21

value: NPC


title: TP-1


title: Batch1_Patient11600-15149_CA11_2

description:
CA11_2Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: Batch1_Patient11600-15149_CA12_1

description:
CA12_1Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: Batch1_Patient11600-15149_CB02_1

description:
CB02_1Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: Control-3

value: Untreated


title: Control-2


title: TG_CD4SP_1


title: Batch1_Patient11600-15149_CA11_1

description:
CA11_1Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: CNES2_ZF91KD_G1A_S19

value: batchC


title: WT_CD4SP_3


title: WT_CD4SP_2


title: WT_CD8SP_3


title: Control-1


title: WT_CD4SP_1


title: Batch1_Patient11600-15149_CA10_2

description:
CA10_2Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: TG_CD4SP_3

value: hCD2-LMO2 knock-in


title: XNES1_ZF91lacZB_S30

value: XDP

value: batchB


title: Batch1_Patient11600-15149_CA10_1

description:
CA10_1Aligned.sortedByCoord.out.bam
counts_batch1.csv

value: Fallopian tube


title: Huh7-LHX2_3

city: Bordeaux

first: Christophe François

last: Grosset

address: 146 rue Léo Saignat

country: France

institute: INSERM

data_processing:
Image analyses and base calling were performed using the HiSeq Control Software and Real-Time Analysis component (Illumina). Demultiplexing was performed using Illumina's conversion software (bcl2fastq 2.20). The quality of the raw data was assessed using FastQC from the Babraham Institute and the Illumina software SAV (Sequencing Analysis Viewer).
A splice junction mapper, TopHat 2.1.1 (using Bowtie 2.3.5.1), was used to align the RNA-Seq reads to the human genome (UCSC GRCh38/hg38) with a set of gene model annotations (genes.gtf downloaded from UCSC on October 27 2019 ; GeneIDs come from the NCBI: gene2refseq.gz).
Final read alignments having more than 3 mismatches were discarded. Samtools (v1.9) was used to sort the alignment files. Then, the counting was performed with Featurecounts 2.0.0. As the data is from a strand-specific assay, the read has to be mapped to the opposite strand of the gene (-s 2 option).
Genome_build: GRCh38/hg38
Supplementary_files_format_and_content: tab-delimited text files include raw gene counts for every gene (Symbol|NCBI GeneID) and every sample

source_name: hepatocellular carcinoma cells

growth_protocol: Huh7 cells were cultured in DMEM 4,5g/l of glucose containing 10% fetal bovine serum and penicillin/streptomycin (1,000 units/mL).

extract_protocol:
Total RNA was isolated using the NucleoSpin RNA Plus (Macherey-Nagel) following manufacturer’s instructions
Libraries were constructed using the Truseq stranded mRNA sample prep kit (Illumina, San Diego, CA,USA) according to the manufacturer instructions.

treatment_protocol: Huh7 cells were transduced with lentiviral particules conteinig LHX2 expression vector (or control vector) and incubated for 48h

value: hepatocellular carcinoma cell line

value: transduced with LHX2 expressing vector

tag: modification

value: LHX2 overexpressiong cells


title: TG_DP_3

value: sorted DP primary thymocytes


title: Batch1_Patient11600-15149_CA09_2

description:
CA09_2Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: TG_DP_2


title: Batch1_Patient11600-15149_CA09_1

description:
CA09_1Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: Batch1_Patient11600-15149_CA08_2

description:
CA08_2Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: XNES1_TRIM28lacZD_S16

tag: individual

value: Control


title: XNES1_TRIM28g1B_S10

tag: dx

value: CRISPRi


title: Huh7-LHX2_2


title: WT_DP_3


title: Huh7-LHX2_1


title: CNES1_ZF91g1D_S20

tag: cell type_at_ko/kd


title: XNES1_ZF91lacZA_S29

tag: condition


title: XNES1_TRIM28lacZC_S15

tag: target gene


title: WT_DP_1


title: CNES2_ZF91KD_G1B_S20

tag: batch


title: XNES1_TRIM28g1A_S9


title: Huh7-CTRL_3

value: transduced with control vector

value: parental cells


title: osnrpd1-1_rep2

city: Beiing

first: Yijun

last: Qi

address: NO.1 Qinghuayuan

department: School of Life Sciences

institute: Tsinghua University

description: osnrpd1-1 replication 2 (sRNA-seq)

library_source: other

data_processing:
sRNA-seq: Cutadapt2.9 was used to find and trim adapter sequences from sRNA-seq reads. 18–-30 nt sequences with adapters trimmed were retained. Clean reads were converted into FASTA format to meet the requirement of miRDeep-P, a software for miRNA identification in plants. The reads in FASTA format were mapped to the rice genome (RGAP7.0) with Bowtie-1.2.3, allowing no mismatches. Small RNA abundance was calculated as reads per million (RPM).  WGBS-seq: Cutadapt2.9 was used to find and trim adapter sequences from Bisulifite-seq reads. Adapters and low-quality reads were removed and clean reads were mapped to the rice genome (RGAP7.0) using BRAT-BW, allowing up to three mismatches. Only uniquely mapped reads were retained. Only uniquely mapped reads were retained. Fractional DNA methylation levels were calculated using BRAT-BW.
Assembly: Oryza sativa RGAP7.0
Supplementary files format and content: bw
Supplementary files format and content: bed

molecule: other

organism: Oryza sativa

source_name: 4-week-old seedling

growth_protocol: Rice plants were grown in a greenhouse at 70% humidity under long-day conditions with daily cycles of 14 h of light at 30°C and 10 h of dark at 25°C. 4-week-old seedlings that start tillering were collected and used for the experiments.

extract_protocol:
sRNA-seq: total RNA was purified from 4-week-old seedlings using TRIzol reagent (Invitrogen, 15596026). Large RNAs were selectively precipitated using 5% polyethylene glycol 8000 (PEG8000; Sigma, P5413) and 0.5 M NaCl (Sigma, S3014). sRNAs were recovered from the supernatant by ethanol precipitation and then separated by electrophoresis using a 15% denaturing PAGE gel. sRNAs that were 18–28 nt long were excised from the gel and purified.  WGBS-seq: genomic DNA was purified from the 4-week-old seedlings using the DNeasy Plant Mini Kit (Qiagen, 69104).
sRNA-seq: purified sRNAs were ligated with the 3’ adaptors and 5’ adaptors. The products were reversely transcribed using SuperScript III Reverse Transcriptase (Invitrogen, 18080093). The cDNAs obtained were amplified by PCR to generate the libraries. The libraries were sequenced on an Illumina HiSeq2500 platform to obtain 50-bp single end reads.   WGBS-seq: Paired-end bisulfite sequencing libraries were constructed using the NEXTflex Bisulfite-Seq kit (Bioo Scientific, Nova-511911). Genomic DNA was sonicated to ~300 bp, end repaired, and 3’ adenylated. The products were ligated with methylated adapters. After size selection with Ampure XP purification beads (Beckman, A63881), the products were subjected to bisulfite conversion by using the EZ DNA Methylation-Gold kit (ZYMO Research, D5005). Bisulfite-converted DNA products were amplified by PCR for 15 cycles using EX Taq DNA polymerase (Takara, RR001C). The PCR products were cleaned up using Ampure XP purification beads (Beckman, A63881). The libraries were validated using the Agilent 2100 Bioanalyzer and sequenced on an Illumina X-Ten platform to obtain 150-bp paired-end reads.

value: 4-week-old seedling


title: CNES1_TRIM28lacZA_S5


title: Huh7-CTRL_1


title: Batch1_Patient11600-15149_CA08_1

description:
CA08_1Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: WT_DP_2

value: wild type


title: TG_DP_1


title: CNES1_TRIM28lacZB_S6

value: NPC


title: Batch1_Patient11600-15149_CA07_2

description:
CA07_2Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: Huh7-CTRL_2


title: osnrpd1-1_rep3

description: osnrpd1-1 replication 3 (sRNA-seq)


title: Batch1_Patient11600-15149_CA07_1

description:
CA07_1Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: Batch1_Patient11600-15149_CA05_2

description:
CA05_2Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: TG_DN_1

value: sorted total DN thymocyte mixture


title: XNES1_TRIM28lacZB_S14

description:
gene_count_matrix_2_xdp.csv
exon_count_matrix_2_xdp.csv
intron32_count_matrix_2_xdp.csv


title: WT_DN_3


title: WT_DN_2


title: CNES1_ZF91g1C_S19


title: TG_DN_3


title: Batch1_Patient11600-15149_CA06_2

description:
CA06_2Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: Batch1_Patient11600-15149_CA06_1

description:
CA06_1Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: osnrpd1-1_rep1

description: osnrpd1-1 replication 1 (sRNA-seq)


title: dcl3a-17_rep2

description: dcl3a-17 replication 2 (sRNA-seq)


title: dcl3a-17_rep3

description: dcl3a-17 replication 3 (sRNA-seq)


title: Batch1_Patient11600-15149_CA05_1

description:
CA05_1Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: CNES1_ZF91g1B_S18


title: TG_DN3_3

value: sorted DN3 primary thymocytes


title: CNES1_TRIM28g1D_S4


title: TG_DN3_2

value: hCD2-LMO2 knock-in


title: Batch1_Patient11600-15149_CA03_1

description:
CA03_1Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: XNES1_ZF91g1D_S28

value: XDP


title: CNES1_ZF91lacZD_S24

value: batchB


title: dcl3a-17_rep1

description: dcl3a-17 replication 1 (sRNA-seq)


title: Batch1_Patient11600-15149_CA04_2

description:
CA04_2Aligned.sortedByCoord.out.bam
counts_batch1.csv

value: Fallopian tube


title: TG_DN_2


title: NIP_rep3

description: Nipponbare replication 3 (sRNA-seq)

value: NIP


title: TG_DN3_1


title: WT_DN_1


title: Batch1_Patient11600-15149_CA04_1

description:
CA04_1Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: XNES1_TRIM28lacZA_S13


title: Batch1_Patient11600-15149_CA03_2

description:
CA03_2Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: NIP_rep2

description: Nipponbare replication 2 (sRNA-seq)


title: CNES1_ZF91lacZC_S23


title: 30_doa10_SM_R

city: Mainz

first: Frank

last: Rühle

address: Ackermannweg 4

department: Bioinformatics Core Facility

country: Germany

institute: Institute of Molecular Biology (IMB)

description: R1, R2, R3, R4, R5 capping and saturation mutagenesis in doa10Δ cells

data_processing:
Demultiplexed fastq files were checked for raw data quality and potential DNA contamination using FastQC (v0.11.9) and FastQScreen (v0.13) software.
Cutadapt (v4.0) was used to trim adapter sequences and for NextSeq-specific 3’ end quality trimming applying a threshold of 20.
Reads were filtered for a minimum length of 15 nucleotides and a maximum expected number of errors equal to 1 computed from quality values using Cutadapt (v4.0).
Read pairs were assembled using the paired-end read merger PEAR (v0.9.11) to reconstruct amplicon sequences based on a minimum of 8 bases overlap of the paired reads. Unassembled read pairs were discarded.
The variable region and the fraction barcode were subsequently extracted from the assembled sequences by applying a design-specific regular expression with the extract command in UMI-Tools (v1.1.2) using flanking common sequences.
Reads were counted for each observed combination of variable region and fraction barcode, and were exported as a count matrix in text format.
Protein stability indices (PSIs) per sequence variant were calculated from count data either per nucleotide sequence or per translated amino acid sequence as described (PMID 29727619) using the analysis pipeline available at: https://gitlab.rlp.net/imbforge/NGSpipe2go/-/tree/MPSprofiling
Assembly: Not applicable, because no alignment applied
Supplementary files format and content: tab delimited text files (tsv) containing counts of each observed combination of variable region (column ‘sequence’) and fraction barcode (column ‘id’).
Library strategy: Multiplexed protein stability (MPS) profiling using counts of amplicon DNA sequences.

source_name: ESM356-1 doa10{delta} yeast strain transformed with the desired plasmid variants

growth_protocol: Yeast cells were grown at 30°C in synthetic complete (SC) medium lacking histidine to log-phase with or without shifting to 37°C before sorting.

extract_protocol:
Yeast cells were lysed by mechanical shearing and then the plasmids therein were isolated by column-purification.
The variable regions were PCR-amplified with the incorporation of unique barcodes for each stability bin. The PCR products were pooled proportionally according to the number of cells sorted into each bin, and then used as the template for another PCR for adding the sample barcodes and illumina sequencing adaptors.

treatment_protocol: Yeast cells were sorted into 8 stability bins according to the tFT readout. The cells sorted into each bin were separately grown to saturation.


title: WT_DN3_2


title: XNES1_ZF91g1C_S27


title: Batch1_Patient11600-15149_CA02_2

description:
CA02_2Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: Batch1_Patient11600-15149_CA02_1

description:
CA02_1Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: NIP_rep1

description: Nipponbare replication 1 (sRNA-seq)


title: CNES1_ZF91g1A_S17


title: WT_DN3_1


title: 27_WT_SM_W

description: W1, W2, W3, Atg1 (-12_-1), Rpa12 (-12_-1) capping and saturation mutagenesis in WT cells

source_name: ESM356-1 yeast strain transformed with the desired plasmid variants


title: CNES1_TRIM28g1C_S3


title: 26_das1_37C_unstable_rep2

description: tFT-X12 degron library in das1Δ cells at 37°C (replicate 2)

source_name: BY4741 das1{delta} yeast strain transformed with the desired plasmid variants


title: Batch1_Patient11600-15149_BH12_2

description:
BH12_2Aligned.sortedByCoord.out.bam
counts_batch1.csv

source_name: Fallopian tube


title: 25_cdc34_37C_unstable_rep2

description: tFT-X12 degron library in cdc34-1 cells at 37°C (replicate 2)

source_name: BY4741 cdc34-1 yeast strain transformed with the desired plasmid variants


title: TG_DN1_3

value: sorted DN1 primary thymocytes


title: TG_DN1_2


title: Batch1_Patient11600-15149_BH12_1

description:
BH12_1Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: XNES1_ZF91g1B_S26


title: TG_DN1_1


title: WT_DN3_3


title: XNES1_TRIM28g1D_S12


title: 28_das1_SM_W

description: W1, W2, W3, Atg1 (-12_-1), Rpa12 (-12_-1) capping and saturation mutagenesis in das1Δ cells

source_name: ESM356-1 das1{delta} yeast strain transformed with the desired plasmid variants


title: WT_DN1


title: 29_WT_SM_R

description: R1, R2, R3, R4, R5 capping and saturation mutagenesis in WT cells

value: ESM356-1 yeast strain transformed with the desired plasmid variants


title: CNES1_ZF91lacZB_S22

description:
gene_count_matrix_2_xdp.csv
exon_count_matrix_2_xdp.csv
intron32_count_matrix_2_xdp.csv

source_name: NPC


title: 23_WT_37C_unstable_rep2

description: tFT-X12 degron library in WT cells at 37°C (replicate 2)

source_name: BY4741 yeast strain transformed with the desired plasmid variants


title: Batch1_Patient11600-15149_CA01_2

description:
CA01_2Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: Batch1_Patient11600-15149_BH11_1

description:
BH11_1Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: CNES1_TRIM28lacZD_S8


title: Batch1_Patient11600-15149_CA01_1

description:
CA01_1Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: 21_das1_unstable_rep3

description: tFT-X12 degron library in das1Δ cells (replicate 3)

value: ESM356-1 das1{delta} yeast strain transformed with the desired plasmid variants


title: XNES1_ZF91lacZD_S32

value: Control


title: 20_WT_unstable_rep3

description: tFT-X12 degron library in WT cells (replicate 3)


title: Batch1_Patient11600-15149_BH09_2

description:
BH09_2Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: 24_cdc53_37C_unstable_rep2

description: tFT-X12 degron library in cdc53-1 cells at 37°C (replicate 2)

source_name: BY4741 cdc53-1 yeast strain transformed with the desired plasmid variants


title: Batch1_Patient11600-15149_BH11_2

description:
BH11_2Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: Tissue culture, SPko-1-input, input, biol rep 2

city: Hannover

first: Maximilian

last: Lenz

address: Carl-Neuberg-Straße 1

department: Institute of Neuroanatomy and Cell Biology

institute: Hannover Medical School

data_processing:
Data analysis was performed at the Galaxy platform (usegalaxy.eu). All files contained more than 10 M high-quality reads (after mapping to the reference genome; mm10) having at least a phred quality of 30 (>90% of total reads).
RNA sequencing data were uploaded to the Galaxy web platform (public server: usegalaxy.eu; (Goecks et al., 2010; Afgan et al., 2018; Galaxy, 2022)) and transcriptome analysis was performed using the Galaxy platform in accordance with the reference-based RNA-seq data analysis tutorial (Batut et al., 2021). Adapter sequencing, low quality, and short reads were removed via the CUTADAPT tool. Reads were mapped using RNA STAR with the mm10 full reference genome (Mus musculus). The evidence-based annotation of the mouse genome (GRCm38), version M25 (Ensembl 100), served as gene model (GENCODE). For an initial assessment of gene expression, unstranded FEATURECOUNT analysis was performed from RNA STAR output. Only samples that contained >60% uniquely mapping reads (feature: “exon”) were considered for further analysis. Statistical evaluation was performed using DESeq2 with “pathway integrity” (ECL experiments) or “coIP” (synaptopodin-coIP experiments) as the primary factor that might affect gene expression. Genes with a low number of mean reads (< 300 BaseMean) were excluded from further analysis. Genes were considered as differentially expressed or enriched if the adjusted p-value was < 0.05. Data visualization was performed according to a modified version of a previously published workflow (Reimand et al., 2019). Further functional enrichment analyses were performed using g:Profiler (version e107_eg54_p17_bf42210) with g:SCS multiple testing correction method applying significance threshold of 0.05 (Raudvere et al., 2019). Gene sets with 100-500 terms were considered for illustration. Heatmaps were generated based on z-scores of the normalized count table. For transcriptome analysis following synaptopodin-coIP, DESeq2 analysis was performed to compare coIP and input samples. Differentially enriched genes in coIP (padj < 0.05 and log2(FC) > 0) were considered for further analyses. To correct for non-specific antibody binding, DESeq2 output (log2(FC)) from the same experimental procedure in synaptopodin-deficient tissue cultures was subtracted from from log2(FC) of differentially enriched genes in wildtype cultures. Only those differentially expressed genes that showed > 30% enrichment in FC after Synpo-KO correction were considered as significantly enriched.
Adapter sequences, low quality, and short reads were removed via the CUTADAPT tool.
Reads were mapped using RNA STAR with the mm10 (Mus musculus) reference genome.
The evidence-based annotation of the mouse genome (GRCm38), version M25 (Ensembl 100), served as gene model (GENCODE). For an initial assessment of gene expression, unstranded FEATURECOUNT analysis was performed from RNA STAR output.
Assembly: mm10
Supplementary files format and content: FEATURE COUNT output (tabular files)

source_name: Brain, organotypic entorhino-hippocampal culture

extract_protocol:
Entorhino-hippocampal tissue cultures were prepared at postnatal day 3-5 from C57BL/6J (wildtype, Synpo+/+) and B6.129-Synpotm1Mndl/Dllr [Synpo-/-, (Deller et al., 2003; Lenz et al., 2021a; Lenz et al., 2021b)] mice of either sex as previously described (Del Turco and Deller, 2007). Synpo-/- strain was back crossed for at least 10 times to establish C57BL/6J genetic background. Cultivation medium contained 50% (v/v) MEM, 25% (v/v) basal medium eagle, 25% (v/v) heat-inactivated normal horse serum, 25 mM HEPES buffer solution, 0.15% (w/v) bicarbonate, 0.65% (w/v) glucose, 0.1 mg/ml streptomycin, 100 U/ml penicillin, and 2 mM glutamax. The pH was adjusted to 7.3. All tissue cultures were allowed to mature for at least 18 days in a humidified atmosphere with 5% CO2 at 35°C. Cultivation medium was replaced 3 times per week.
Synpo-coIP: Six tissue cultures per biological replicate were washed in coIP-solution and dounce homogenized on ice in a solution containing (150 µl per sample, coIP-solution, in mM): 10 HEPES, 120 NaCl, 0.05% (v/v) NP-40 or IGEPAL CA-630 (Sigma Aldrich, #I8896), cOmplete protease inhibitor (Roche, according to manufacturer’s instructions), murine RNase inhibitor (1:1000, New England Biolabs, #M0314) and cycloheximide (0.5 mg/ml, Sigma Aldrich, #C-4859). Input was precleared twice with protein A beads (10 µl per 100 µl sample/supernatant) to reduce non-specific binding. Protein A beads (10 µl, New England Biolabs, #S1425) were washed twice in coIP-solution and consecutively incubated for 1 hour at 4°C with an anti-synaptopodin antibody (rabbit anti-Synaptopodin, Synaptic Systems, #163002). Beads were carefully washed twice to remove unbound antibodies and incubated for 1 hour with the precleared supernatant (‘input’) at 4 °C with head-over-tail rotation. 10 µl of the precleared supernatant were kept as input control. After incubation, beads were washed twice with coIP-solution and finally dissolved in RNA protection buffer (New England Biolabs). RNA was released from the beads through protein K lysis (Monarch® Total RNA Miniprep Kit; New England Biolabs, #T2010S). After lysis, beads were removed from the solution in a magnetic rack and the RNA containing supernatant was transferred to fresh tubes. RNA isolation on coIP- and input-samples was then performed according to the manufacturer’s instructions. RNA content was determined using the Agilent RNA 6000 Pico Kit (Agilent, #5067-1513) with a 2100 Bioanalyzer (Agilent, #G2939BA). RNA Library preparations for Synpo-related transcriptome analysis were performed using the NEBNext® Single Cell/Low Input RNA Library Prep Kit for Illumina® (New England Biolabs, #E6420) according to the manufacturer's instructions. We quantified the libraries using the NEBNext Library Quant Kit for Illumina (New England Biolabs, #E7630) based on the mean insert size provided by the Bioanalyzer. A 10 nM sequencing pool (120 µl in Tris-HCl, pH 8.5) was generated for sequencing on the NovaSeq6000 Sequencing platform (Illumina; service provided by CeGaT GmbH, Tübingen, Germany). We performed a paired-end sequencing with 150 bp read length. Regional mRNA library preparation: RNA Library preparations for transcriptome analysis were performed using the NEBNext® Single Cell/Low Input RNA Library Prep Kit for Illumina® (New England Biolabs, #E6420) according to the manufacturer's instructions. Briefly, isolation of the dentate gyrus and CA3 area from individual tissue cultures was performed using a scalpel. One isolated dentate gyrus or CA3 area respectively was transferred to 7.5 µl lysis buffer (supplemented with murine RNase inhibitor) and homogenized using a pestle. Samples were centrifuged for 30 s at 10,000 g and 5 µl of supernatant were collected from individual samples and further processed. After cDNA synthesis, cDNA amplification was performed according to the manufacturer's protocol with 12 PCR cycles. cDNA yield was consecutively analyzed by a High Sensitivity DNA assay on a Bioanalyzer instrument (Agilent). cDNA amount was adjusted to 10 ng for further downstream applications. After fragmentation and adaptor ligation, dual index primers (New England Biolabs, #E7600S) were ligated in a library amplification step using 10 PCR cycles. Libraries were finally cleaned up with 0.8X SPRI beads following a standard bead purification protocol. Library purity and size distribution was assessed with a High Sensitivity DNA assay on a Bioanalyzer instrument (Agilent). We quantified the libraries using the NEBNext Library Quant Kit for Illumina (New England Biolabs, #E7630) based on the mean insert size provided by the Bioanalyzer. A 10 nM sequencing pool (120 µl in Tris-HCl, pH 8.5) was generated for sequencing on the NovaSeq6000 Sequencing platform (Illumina; service provided by CeGaT GmbH, Tübingen, Germany). We performed a paired-end sequencing with 100 bp read length.

value: synaptopodin-deficient

tag: fraction

value: input


title: Tissue culture, SPko-2-input, input, biol rep 1

value: Brain, organotypic entorhino-hippocampal culture


title: Batch1_Patient11600-15149_BH09_1

description:
BH09_1Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: XNES1_ZF91g1A_S25

value: CRISPRi


title: XNES1_ZF91lacZC_S31


title: XNES1_TRIM28g1C_S11


title: Tissue culture, SPko-2-coIP, Synpo-coIP, biol rep 2

value: Synpo-coIP


title: 17_doa10_unstable_rep1

description: tFT-X12 degron library in doa10Δ cells (replicate 1)

value: ESM356-1 doa10{delta} yeast strain transformed with the desired plasmid variants


title: Batch1_Patient11600-15149_BH08_2

description:
BH08_2Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: 19_das1_Cterminome_rep2

description: yeast C-terminome library in das1Δ cells (replicate 2)


title: 18_WT_Cterminome_rep2

description: yeast C-terminome library in WT cells (replicate 2)


title: Batch1_Patient11600-15149_BH08_1

description:
BH08_1Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: CUTTAGiTregRestim_1

city: Beijing

first: Huiyun

last: Lyu

address: Tsinghua University, Zhengyutong Building D301

department: Institute for Immunology

data_processing:
RNA-seq analysis: Raw fastq reads were trimmed by Cutadapt (version 1.18) to trim adapter and low quality sequence.  Reads were aligned to the mouse genome (mm10) using STAR (version 2.5.3). The number of reads within each gene in each single cell were counted using RSEM (version 1.3.0) with gene annotation file from GENCODE (GRCm38.m23).  Differential expression was estimated by using DESeq2 package (version 1.26) with absolute log2 Fold change > 0.5 and adjusted P value < 0.05. Functional enrichment analysis were performed by using the clusterProfiler R package (v3.14.3) with default parameters (P value < 0.05) and the functional annotations terms in Gene Ontology (GO)
ATAC-seq analysis: Raw fastq reads were trimmed by Cutadapt (version 1.18) to trim adapter and low quality sequence.  Reads were aligned to the mouse genome (mm10) using Bowtie2 (version 2.3.3.1), the parameters are -t -q -N 1 -L 25 -X 2000 --no-mixed --no-discordant. Reads with alignment quality < Q30, mapped to chrM, overlapping with ENCODE blacklisted regions ) were discarded. Duplicates were removed using Picard (version 2.20.4) and Samtools (version 1.13). Open chromatin region peaks were called on using MACS2 peak caller (version 2.2.7.1) with the following parameters: -nomodel -nolambda -call-summits. Peaks from all samples were merged and peaks falls in ENCODE blacklisted regions were filtered out.  Differential peaks were estimated by using “edgeR” method in the R package DiffBind (version 2.14.0) with absolute log2 Fold change > 1 and adjusted P value < 0.05. The peaks annotations were performed with the “annotatePeak” function in the R package ChIPseeker52. The plot of ATAC-seq signals over a set of genomic regions were calculated by using “computeMatrix” function in deepTools (version 2.0) and plotted by using “plotHeatmap” and “plotProfile” functions in deepTools.
Cut&Tag data analysis Raw fastq reads were trimmed by Cutadapt (version 1.18) to trim adapter and low quality sequence.  Reads were aligned to the mouse genome (mm10) using Bowtie2 (version 2.3.3.1), the parameters are -t -q -N 1 -L 25 -X 2000 --no-mixed --no-discordant.  Reads with alignment quality < Q30, mapped to chrM, overlapping with ENCODE blacklisted regions (https://sites.google.com/site/anshulkundaje/projects/blacklists) were discarded. Duplicates were removed using Picard (version 2.20.4) and Samtools (version 1.13). Open chromatin region peaks were called using MACS2 peak caller (version 2.2.7.1) with the following parameters: -nomodel -nolambda -call-summits. Peaks from all samples were merged and peaks falls in ENCODE blacklisted regions were filtered out.  The peaks annotations were performed with the “annotatePeak” function in the R package ChIPseeker52. The plot of Cut&Tag signals over a set of genomic regions were calculated by using “computeMatrix” function in deepTools and plotted by using “plotHeatmap” and “plotProfile” functions in deepTools. The motif enrichment analysis was performed by using the "findMotifsGenome.pl" command in HOMER with default parameters. The motif occurrences in each peak were identified by using FIMO (MEME suite v5.0.4) with the following settings: a first-order Markov background model, a P value cutoff of 10-4, and PWMs from the mouse HOCOMOCO motif database (v11).
Assembly: mm10
Library strategy: CUT&Tag

source_name: iTreg

extract_protocol:
Live cells were sorted using PI staining, then 5 × 104 cells were transferred to a low-binding centrifuge tube and resuspended in 50 μL of pre-chilled Lysis Buffer. After 20 times pipetting, cells were incubated on ice for 10 minutes for lysis.
RNA-seq The RNA-seq library preparation was performed by a modified SMART-seq2 protocol49,51. Briefly, RNA was added into 4 µl elution mix made of 1 µl RT primer (10 µM), 1 µl dNTP mix (10 mM each), 1 µl RNase inhibitor (4 U/µl), and 1 µl H2O. Eluted samples were incubated at 72 °C for 3 min and immediately placed on ice. Each sample was added with 7 µl reverse transcription (RT) mix made of 0.75 µl H2O, 0.1 µl Maxima RNase-minus RT (Thermo Fisher Scientific, #EP0741), 2 µl 5x Maxima RT buffer, 2 µl Betaine (5 M, Sigma-Aldrich, B0300), 0.9 µl MgCl2 (100 mM), 1 µl TSO primer (10 µM), 0.25 µl RNase inhibitor (40 U/µl).The RT reaction was incubated at 42 °C for 90 min and followed by 10 cycles of (50 °C for 2 min, 42 °C for 2 min), then heat inactivated at 70 °C for 15 min. Samples were then amplified with an addition of 14 µl PCR mix made of 1 µl H2O, 0.5 µl ISPCR primer (10 µM), 12.5 µl KAPA HiFi HotStart ReadyMix (KAPA Biosystems, KK2602). The PCR reaction was performed as follows: 98 °C for 3 min, 22 cycles of (98 °C for 15 sec, 67 °C for 20 sec, 72 °C for 6 min), and final extension at 72 °C for 5 min.  The amplified cDNA product was purified using VAHTS DNA Clean Beads. Sequencing libraries were prepared using TruePrep DNA Library Prep Kit V2 for Illumina (Vazyme, TD503-02).  Primer sequences: RT primer (Sangon), 5’Biotin-AAGCAGTGGTATCAACGCAGAGTACTTTTTT TTTTTTTTTTTTTTTTTTTTTTTTVN; TSO primer (Sangon), 5’Biotin-AAGCAGTGGTATCAAC CAGAGTACAT/rG//rG//iXNA_G/; ISPCR primer (Sangon), 5’Biotin-AAGCAGTGGTATCAACGC AGA*G*T. ATAC-sequencing ATAC-seq were performed using TruePrep DNA Library Prep Kit V2 for Illumina according to the manufacturer’s recommendation. Briefly, live cells were sorted using PI staining, then 5 × 104 cells were transferred to a low-binding centrifuge tube and resuspended in 50 μL of pre-chilled Lysis Buffer. After 20 times pipetting, cells were incubated on ice for 10 minutes for lysis. Subsequently, cells were pelleted by centrifugation at 4°C 500 g for 5 minutes, and suspended in a mixture consisting of 10 μL of 5× TTBL, 5 μL of TTE Mix V50, and 35 μL of ddH2O. The mixture was gently mixed by pipetting 20 times, and incubated at 37°C for 30 minutes for fragmentation. The products were purified using 2× VAHTS DNA Clean Beads, eluted with 26 μL of water, and collected for the subsequent amplification. 24 μl of the eluted samples were amplified with different combination of i7 index and i5 index primers. QPCR were performed to determine the cycle number for PCR amplification.0.55×VAHTS DNA Clean Beads was employed for size selection of the PCR product. Finally, 30 ng of each sample were pooled for subsequent second-generation sequencing, on the Illumina NovaSeq 6000 with read length of 150 bp.  Cut&Tag assay Foxp3-RFP+ iTreg were induced, and infected with NFAT-Flag-GFP lentiviruses on day 1.5 and day 2.5 of induction. On day 5, Foxp3-RFP+ MSCV-NFAT-Flag-GFP+ iTregs were sorted, then rested and restimulated for 2 day. Cut&Tag assay were performed using Hyperactive Universal CUT&Tag Assay Kit for Illumina (Vazyme Biotech Co.,Ltd) according to the manufacturer’s recommendation. Briefly, 1 × 105 iTregs were collected to low-binding centrifuge tubes and washed. Nucleus isolation were performed by NE buffer for 10 min on ice, and cross-linked by 0.1% formaldehyde for 2 min, then stopped by glycine. Con A Beads were pre-treated with binding buffer, and then incubated with nuclei of iTreg for 10 min. Further, samples were incubated with anti-Flag M2 antibody (1:50) or without primary antibody (Mock) overnight at 4°C. Goat Anti-Mouse IgG secondary antibody (1:50) was added and incubated for 1 h. Cells were washed three times, and then incubated with pA/G-Tnp enzyme for 1 h. After incubation, samples were mixed with 5×TTBL and Dig-300 Buffer and incubated at 37°C for 60 min for fragmentation. DNA was extracted and purified, then amplified with different combination of i7 index and i5 index primers. QPCR was performed to determine the cycle number for PCR amplification.VAHTS DNA Clean Beads were employed for size selection of the PCR product. Finally, 30 ng of each sample were pooled for subsequent second-generation sequencing, on the Illumina NovaSeq 6000 with read length of 150 bp.

value: iTreg

tag: antibody

value: anti-Flag M2


title: CUTTAGiTregRestim_2


title: Batch1_Patient11600-15149_BH10_2

description:
BH10_2Aligned.sortedByCoord.out.bam
counts_batch1.csv

data_processing:
Paired-end reads from Illumina NextSeq 500 were processed and mapped using STAR
The following filters were used to eliminate cells with (1) UMI counts < 10,000 and (2) gene counts < 200
Removed genes detected in < 3 cells
Assembly: UCSC hg19
Supplementary files format and content: Gene-cell matrix table containing transcript counts for every gene in every cell for each sample in batch1
Supplementary files format and content: Gene-cell matrix table containing transcript counts for every gene in every cell for each sample in batch2


title: CNES1_ZF91lacZA_S21

description:
gene_count_matrix_2_xdp.csv
exon_count_matrix_2_xdp.csv
intron32_count_matrix_2_xdp.csv

value: NPC


title: Batch1_Patient11600-15149_BH10_1

description:
BH10_1Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: Tissue culture, SPko-1-coIP, Synpo-coIP, biol rep 1


title: CNES1_TRIM28lacZC_S7


title: 22_doa10_unstable_rep2

description: tFT-X12 degron library in doa10Δ cells (replicate 2)


title: CNES1_TRIM28g1B_S2


title: CUTTAGiTregRest_2


title: 16_das1_unstable_rep2

description: tFT-X12 degron library in das1Δ cells (replicate 2)


title: Batch1_Patient11600-15149_BH07_2

description:
BH07_2Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: 15_WT_unstable_rep2

description: tFT-X12 degron library in WT cells (replicate 2)


title: Batch1_Patient11600-15149_BH07_1

description:
BH07_1Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: CNES1_TRIM28g1A_S1


title: CUTTAGiTregRest_1


title: CUTTAGiTregMock_2


title: 14_das1_37C_unstable_rep1

description: tFT-X12 degron library in das1Δ cells at 37°C (replicate 1)

value: BY4741 das1{delta} yeast strain transformed with the desired plasmid variants


title: CNES3_ZF91lacZD_S30

value: batchA


title: Batch1_Patient11600-15149_BH06_2

description:
BH06_2Aligned.sortedByCoord.out.bam
counts_batch1.csv

value: Fallopian tube


title: CNES3_ZF91lacZC_S29


title: 13_cdc34_37C_unstable_rep1

description: tFT-X12 degron library in cdc34-1 cells at 37°C (replicate 1)

value: BY4741 cdc34-1 yeast strain transformed with the desired plasmid variants


title: CNES3_ZF91lacZB_S28


title: CUTTAGiTregMock_1


title: Batch1_Patient11600-15149_BH05_2

description:
BH05_2Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: ATACnTregRest_2

data_processing:
RNA-seq analysis: Raw fastq reads were trimmed by Cutadapt (version 1.18) to trim adapter and low quality sequence.  Reads were aligned to the mouse genome (mm10) using STAR (version 2.5.3). The number of reads within each gene in each single cell were counted using RSEM (version 1.3.0) with gene annotation file from GENCODE (GRCm38.m23).  Differential expression was estimated by using DESeq2 package (version 1.26) with absolute log2 Fold change > 0.5 and adjusted P value < 0.05. Functional enrichment analysis were performed by using the clusterProfiler R package (v3.14.3) with default parameters (P value < 0.05) and the functional annotations terms in Gene Ontology (GO)
ATAC-seq analysis: Raw fastq reads were trimmed by Cutadapt (version 1.18) to trim adapter and low quality sequence.  Reads were aligned to the mouse genome (mm10) using Bowtie2 (version 2.3.3.1), the parameters are -t -q -N 1 -L 25 -X 2000 --no-mixed --no-discordant. Reads with alignment quality < Q30, mapped to chrM, overlapping with ENCODE blacklisted regions ) were discarded. Duplicates were removed using Picard (version 2.20.4) and Samtools (version 1.13). Open chromatin region peaks were called on using MACS2 peak caller (version 2.2.7.1) with the following parameters: -nomodel -nolambda -call-summits. Peaks from all samples were merged and peaks falls in ENCODE blacklisted regions were filtered out.  Differential peaks were estimated by using “edgeR” method in the R package DiffBind (version 2.14.0) with absolute log2 Fold change > 1 and adjusted P value < 0.05. The peaks annotations were performed with the “annotatePeak” function in the R package ChIPseeker52. The plot of ATAC-seq signals over a set of genomic regions were calculated by using “computeMatrix” function in deepTools (version 2.0) and plotted by using “plotHeatmap” and “plotProfile” functions in deepTools.
Cut&Tag data analysis Raw fastq reads were trimmed by Cutadapt (version 1.18) to trim adapter and low quality sequence.  Reads were aligned to the mouse genome (mm10) using Bowtie2 (version 2.3.3.1), the parameters are -t -q -N 1 -L 25 -X 2000 --no-mixed --no-discordant.  Reads with alignment quality < Q30, mapped to chrM, overlapping with ENCODE blacklisted regions (https://sites.google.com/site/anshulkundaje/projects/blacklists) were discarded. Duplicates were removed using Picard (version 2.20.4) and Samtools (version 1.13). Open chromatin region peaks were called using MACS2 peak caller (version 2.2.7.1) with the following parameters: -nomodel -nolambda -call-summits. Peaks from all samples were merged and peaks falls in ENCODE blacklisted regions were filtered out.  The peaks annotations were performed with the “annotatePeak” function in the R package ChIPseeker52. The plot of Cut&Tag signals over a set of genomic regions were calculated by using “computeMatrix” function in deepTools and plotted by using “plotHeatmap” and “plotProfile” functions in deepTools. The motif enrichment analysis was performed by using the "findMotifsGenome.pl" command in HOMER with default parameters. The motif occurrences in each peak were identified by using FIMO (MEME suite v5.0.4) with the following settings: a first-order Markov background model, a P value cutoff of 10-4, and PWMs from the mouse HOCOMOCO motif database (v11).
Assembly: mm10

source_name: nTreg

value: nTreg


title: 11_WT_37C_unstable_rep1

description: tFT-X12 degron library in WT cells at 37°C (replicate 1)

value: BY4741 yeast strain transformed with the desired plasmid variants


title: 12_cdc53_37C_unstable_rep1

description: tFT-X12 degron library in cdc53-1 cells at 37°C (replicate 1)

value: BY4741 cdc53-1 yeast strain transformed with the desired plasmid variants


title: Batch1_Patient11600-15149_BH04_2

description:
BH04_2Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: CNES3_ZF91g1D_S26

value: Control


title: ATACnTregRest_1


title: CNES3_ZF91lacZA_S27


title: 10_das1_Cterminome_rep1

description: yeast C-terminome library in das1Δ cells (replicate 1)

value: ESM356-1 das1{delta} yeast strain transformed with the desired plasmid variants


title: 09_WT_Cterminome_rep1

description: yeast C-terminome library in WT cells (replicate 1)

value: ESM356-1 yeast strain transformed with the desired plasmid variants


title: ATACnTregAct_2


title: ATACnTregAct_1


title: Batch1_Patient11600-15149_BH04_1

description:
BH04_1Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: Batch1_Patient11600-15149_BH05_1

description:
BH05_1Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: Batch1_Patient11600-15149_BH03_2

description:
BH03_2Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: CNES3_ZF91g1C_S25

description:
gene_count_matrix_2_xdp.csv
exon_count_matrix_2_xdp.csv
intron32_count_matrix_2_xdp.csv


title: CNES3_ZF91g1B_S24


title: Batch1_Patient11600-15149_BH06_1

description:
BH06_1Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: ATACTcRest_1

source_name: Tconv

value: Tconv


title: ATACTcRest_2


title: 07_das1_SM

description: IN1, IN2, LN1, LN3, VN2 saturation mutagenesis in das1Δ cells


title: Batch1_Patient11600-15149_BH03_1

description:
BH03_1Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: 08_Cdeg_12X_rep2

description: tFT-X12 library (replicate 2)


title: CNES3_ZF91g1A_S23

value: CRISPRi


title: 06_WT_SM

description: IN1, IN2, LN1, LN3, VN2 saturation mutagenesis in WT cells


title: Batch1_Patient11600-15149_BH02_2

description:
BH02_2Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: 05_das1_cap

description: IN1, IN2, LN1, LN3, VN2 capping in das1Δ cells


title: ATACTcAct_2


title: CNES3_TRIM28lacZD_S15


title: Batch1_Patient11600-15149_BH02_1

description:
BH02_1Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: CNES3_TRIM28lacZC_S14


title: ATACTcAct_1


title: 04_WT_cap

description: IN1, IN2, LN1, LN3, VN2 capping in WT cells


title: Batch1_Patient11600-15149_BH01_2

description:
BH01_2Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: CNES3_TRIM28lacZB_S13


title: Batch1_Patient11600-15149_BH01_1

description:
BH01_1Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: 03_das1_unstable_rep1

description: tFT-X12 degron library in das1Δ cells (replicate 1)


title: ATACiTregRest_2


title: Batch1_Patient11600-15149_BG12_2

description:
BG12_2Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: CNES3_TRIM28g1D_S11


title: CNES3_TRIM28lacZA_S12


title: 01_Cdeg_12X_rep1

description: tFT-X12 library (replicate 1)


title: ATACiTregRest_1


title: SS2nTregRest_3


title: Batch1_Patient11600-15149_BG12_1

description:
BG12_1Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: SS2nTregRest_2


title: CNES3_TRIM28g1C_S10


title: CNES3_TRIM28g1B_S9


title: SS2nTregRest_1


title: Batch1_Patient11600-15149_BG11_2

description:
BG11_2Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: CNES3_TRIM28g1A_S8


title: 231-LM-2

city: Chongqing

first: Shaojie

last: Cheng

state: -- None selected --

address: No.1, Yi-Xue-Yuan Road, Yu-Zhong District

institute: Chongqing Medical University

data_processing:
Quality control: The quality of raw reads was first assessed using the FastQC. Raw data (raw reads) of fastq format were firstly processed through in-house perl scripts. In this step, clean data (clean reads) were obtained by removing reads containing adapter, reads containing ploy-N and low quality reads from raw data.
Genomic alignment and quantification: Reads were mapped to human genome assembly GRCh38 (ftp://ftp.ensembl.org/pub/release-89/fasta/homo_sapiens/dna/Homo_sapiens.GRCh38.dna.toplevel.fa.gz) or mouse genome assembly GRCm38 (ftp://ftp.ensembl.org/pub/release-102/fasta/mus_musculus/Mus_musculus.GRCm38.dna.toplevel.fa.gz) using HISAT2. HTSeq v0.13.5 was used to count the reads numbers mapped to each gene. And then FPKM of each gene was calculated based on the length of the gene and reads count mapped to this gene. Human GTF file:https://ftp.ensembl.org/pub/release-104/gtf/homo_sapiens/Homo_sapiens.GRCh38.104.gtf.gz. Mmouse GTF file:https://ftp.ebi.ac.uk/pub/databases/gencode/Gencode_mouse/release_M20/gencode.vM20.annotation.gtf.gz.
DESeq2 (v2) was applied for the detection of differentially expressed mRNAs between tumor and non-tumor samples, the limma R package (3.40.6) was used for differentially expressed circRNAs.
Assembly: GRCh38, GRCm38
Supplementary files format and content: tab-delimited text files include RPKM values for each Sample

source_name: MDA-MB-231 cell

growth_protocol: 4T1 cells were cultured in RPMI 1640 medium (Gibco, USA) with 10% fetal bovine serum (FBS) (HyClone) and 1% penicillin/streptomycin (Beyotime, Shanghai, China). MDA-MB-231 cells were cultured in DMEM medium (Gibco, USA) with 10% FBS and 1% penicillin/streptomycin. All cell lines were cultured in a 5% CO2 incubator at 37°C.

extract_protocol:
RNA was extracted using TRIzol reagent (Invitrogen). 1.3 ug of total RNA was used for the construction of sequencing libraries.
A total amount of 3 μg RNA per sample was used as input material for the RNA sample preparations. Firstly,ribosomal RNA was removed by Epicentre Ribo-zero™ rRNA Removal Kit (Epicentre, USA), and rRNA free residue was cleaned up by ethanol precipitation. Subsequently, sequencing libraries were generated using the rRNA-depleted RNA by NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer’s recommendations.

value: MDA-MB-231 cell

value: TNBC

value: LM


title: 231-LM-3


title: Batch1_Patient11600-15149_BG11_1

city: Oxford

first: Ahmed

last: Ahmed

address: John Radcliffe Hospital, Headley way

description:
BG11_1Aligned.sortedByCoord.out.bam
counts_batch1.csv

extract_protocol:
Isolated WβA or non-WβA cells were sorted into 96-well plates containing 4 μl lysis buffer supplemented with 0.1 μl RNase inhibitor (Clonetech), 1.9 μl 0.4% Triton X-100, 1 μl 10 μM 5‘-biotinylated oligo-dT30VN (IDT) and 1 μl 10 mM dNTP (Thermo Scientific). Cells were sorted at one cell per well, with bulk controls (10 cells) and empty well controls (0 cells) included for each plate. Plates were snap frozen on dry ice and stored at - 80 degrees C for less than 4 weeks.
Single cell cDNA synthesis and library generation were performed according to the SMART-seq2 protocol (Picelli et al., 2014), as previously described (Hu et al., 2020). Briefly, cells were lysed by removing plates from  -80 0C and heating at 72 0C for 3 min. Plates were then placed at 4 0C before adding the reverse transcription mix containing 5’-biotinylated TSO (Qiagen). PCR products were cleaned up using 0.8:1 Ampure XP beads (Beckman Coulter) with Biomek FxP Laboratory Automation Workstation (Biomek). Quality of single-cell cDNA was tested using TapeStation, as well as by single cell qPCR for GAPDH or ACTB using the QuantiNova SYBR Green PCR Kit (Qiagen). cDNA concentration was measured using Quant-iTTM PicoGreenTM dsDNA Assay Kit (Invitrogen) on the CLARIOstar Plate Reader (BMG Labtech). Wells with CT values of GAPDH or ACTB below 20 were selected as wells with good quality cDNA. Libraries from single cell cDNA were generated using miniaturized Nextera XT (Illumina) protocol (Mora-Castilla et al., 2016) with Mosquito HTS (TTP LabTech), in 384-well Endure plate (Life Technology). Library sequencing was performed by Novogene.


title: Batch1_Patient11600-15149_BG10_2

description:
BG10_2Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: SS2nTregAct_2


title: XNES2_ZF91lacZD_S40

description:
gene_count_matrix_2_xdp.csv
exon_count_matrix_2_xdp.csv
intron32_count_matrix_2_xdp.csv

value: XDP


title: SS2nTregAct_3


title: XNES2_ZF91lacZC_S22

value: NPC


title: 231-LM-1


title: Batch1_Patient11600-15149_BG10_1

description:
BG10_1Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: 02_WT_unstable_rep1

description: tFT-X12 degron library in WT cells (replicate 1)


title: 231-Pri-3

value: Pri


title: SS2TcRest_3


title: Batch1_Patient11600-15149_BG09_2

description:
BG09_2Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: XNES2_ZF91lacZB_S21

value: batchA


title: 231-Pri-1


title: SS2TcRest_2


title: Batch1_Patient11600-15149_BG09_1

description:
BG09_1Aligned.sortedByCoord.out.bam
counts_batch1.csv

value: Fallopian tube


title: XNES2_ZF91lacZA_S20


title: 231-Pri-2


title: SS2TcRest_1


title: Batch1_Patient11600-15149_BG08_2

description:
BG08_2Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: XNES2_ZF91g1D_S19


title: Donor 9429 upregulated GIRK2, alcohol-naïve, batch b, bio rep 2

city: New York

first: Paul

last: Slesinger

address: 1425 Madison Ave

department: Neuroscience

institute: Icahn School of Medicine at Mount Sinai

data_processing:
Raw reads were assessed for quality using FastQC (version 0.11.8) before and after adapter sequence trimming by Cutadapt (version 4.1).
Reads were pseudoaligned using Salmon (version 0.13.1) for estimation of normalized gene expression (TPM, i.e. transcript per million).
Reads were also aligned to the human reference genome (Gencode version 28) and quantified for gene read counts, both in STAR (version 2.5.3a).
For differential gene expression analyses, read counts were modelled in the linear mixed model using batch, sex and donor information as the random effect terms by the R (version 4.1.0) package "variancePartition" (version 1.28.0).
Assembly: hg38
Supplementary files format and content: "star_gene.tsv" includes reads counts for genes (in rows) across samples (in columns)
Supplementary files format and content: salmon_gene.tsv includes TPM values for genes (in rows) across samples (in columns)

growth_protocol: Doxycycline-inducible transcription factor NGN2 with neuomycin selection and rt-TA lentiviruses were used on neural progenitor cells. After dox-induction and antibiotic selection, cells were grown in Neurobasal medium with B27 supplement up to 14 days post-induction, then transitioned to Neurobasal Plus medium.

extract_protocol:
Total RNA was extracted from fresh frozen cell pellet samples using Qiagen RNeasy Plus Universal mini kit following manufacturer’s instructions
RNA sequencing libraries were prepared using the NEBNext Ultra RNA Library Prep Kit for Illumina using manufacturer’s instructions

treatment_protocol: 17 mM ethanol exposure began at 21 days post-induction, through harvest at 42 days post-induction. EtOH was supplemented daily to account for evaporation.

value: NGN2 neurons

value: Naïve

tag: upregmethod

value: Lenti


title: Donor 9429 upregulated GIRK2, alcohol-naïve, batch b, bio rep 3

tag: cell type


title: SS2TcAct_3


title: Donor 9429 upregulated GIRK2, alcohol-naïve, batch b, bio rep 1

tag: treatment


title: SS2nTregAct_1

value: nTreg


title: Batch1_Patient11600-15149_BG07_2

institute: University of Oxford

description:
BG07_2Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: Donor 9429 upregulated GIRK2, alcohol-naïve, batch a, bio rep 3


title: SS2TcAct_2


title: XNES2_ZF91g1B_S17


title: XNES2_ZF91g1A_S16


title: Donor 9429 upregulated GIRK2, alcohol-naïve, batch a, bio rep 2


title: Batch1_Patient11600-15149_BG06_2

description:
BG06_2Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: XNES2_TRIM28lacZD_S7

value: Control


title: Donor 9429 upregulated GIRK2, alcohol-naïve, batch a, bio rep 1


title: Batch1_Patient11600-15149_BG06_1

description:
BG06_1Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: XNES2_TRIM28lacZC_S6


title: SS2TcAct_1


title: XNES2_ZF91g1C_S18


title: XNES2_TRIM28lacZB_S5


title: Donor 9429 upregulated GIRK2, alcohol-chronic, batch b, bio rep 3

value: Chronic


title: Batch1_Patient11600-15149_BG05_2

description:
BG05_2Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: Batch1_Patient11600-15149_BG05_1

description:
BG05_1Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: SS2iTregRestim_3

value: iTreg


title: XNES2_TRIM28lacZA_S4


title: SS2iTregRestim_2


title: SS2iTregRest_3


title: SS2iTregRestim_1


title: Donor 9429 upregulated GIRK2, alcohol-chronic, batch b, bio rep 2


title: Batch1_Patient11600-15149_BG08_1

description:
BG08_1Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: Donor 9429 upregulated GIRK2, alcohol-chronic, batch b, bio rep 1


title: SS2iTregRest_2


title: Batch1_Patient11600-15149_BG04_2

description:
BG04_2Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: XNES2_TRIM28g1D_S39


title: XNES2_TRIM28g1C_S3


title: Batch1_Patient11600-15149_BG07_1

description:
BG07_1Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: Batch1_Patient11600-15149_BG04_1

description:
BG04_1Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: SS2iTregRest_1


title: Donor 9429 upregulated GIRK2, alcohol-chronic, batch a, bio rep 3


title: Donor 9429 upregulated GIRK2, alcohol-chronic, batch a, bio rep 2


title: Batch1_Patient11600-15149_BG03_2

description:
BG03_2Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: XNES2_TRIM28g1A_S2


title: SS2iTregORAIinh_3


title: XNES2_TRIM28g1A_S1


title: Donor 9429 endogenous GIRK2, alcohol-naïve, batch b, bio rep 3

value: isoCTL


title: Donor 9429 upregulated GIRK2, alcohol-chronic, batch a, bio rep 1


title: Batch1_Patient11600-15149_BG02_2

description:
BG02_2Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: Batch1_Patient11600-15149_BG03_1

description:
BG03_1Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: Donor 9429 endogenous GIRK2, alcohol-naïve, batch b, bio rep 2


title: subq,MRTX,BFPpos,HTO

first: ZHUXUAN

last: LI

address: 417 E 68th St

institute: Memorial Sloan Kettering Cancer Center

data_processing:
FASTQ files of single-cell RNA-sequencing data generated on the 10X Chromium platform were processed using the standard CellRanger pipeline (version 5.0.0). Reads were aligned to a custom GRCm38 / mm10 reference including the tdTomato, GFP, and mKate transgenes used in this study. Cell-gene count matrices were analyzed using a combination of published packages and custom scripts centered around the scanpy / AnnData ecosystem. Single-cell RNA-sequencing datasets from GFPshRNA KP LUAD tumors, MRTX1133-treated KP LUAD tumors, and lung regeneration experiments were analyzed separately using the same workflows.  Single-cell RNA-sequencing data from treated and control replicates were dehashed and compiled into a combined count matrix. Cells with less than 500 UMIs, more than 20% mitochondrial UMIs, and low complexity based on the number of detected genes vs. number of UMIs were removed. Doublets were filtered using scrublet. UMI counts were normalized using the size factor approach described by Lun et al. .  Highly variable features were selected using a variance stabilizing transformation and dimensionality reduction was performed on normalized, log2-transformed count data using principal component analysis. The dimensionality reduced count matrices were used as input for UMAP-embedding and unsupervised clustering with the leiden algorithm; bbknn was used to control for batch effects.
Supplementary files format and content: mm10
Supplementary files format and content: matrix files

source_name: subcutanous tumors

extract_protocol:
To dissociate tumors into single-cell suspensions, primary tumors were finely chopped with scissors and incubated with digestion buffer containing collagenase IV (#17104019, ThermoFisher Scientific, 0.1 U/ml), dispase (#354235, Corning, 0.6 U/ml), and DNase I (#69182–3; Sigma Aldrich, 10 U/ml). For dissociating normal lung epithelial cells, digestion buffer was administrated intratracheally into the lung as described before (27). Following enzymatic dissociation, samples were washed with 2% heat-inactivated FBS in S-MEM (#11380037, Thermo Fisher Scientific), filtered through a 100 μm cell strainer (#431752, Corning), and centrifuged at 1500 rpm for 5 min at 4 °C. The supernatant was removed, and the pellet was resuspended in lysis buffer (#555899, BD Biosciences) to remove red blood cells. Cells were passed through a 40 μm strainer (#431750, Corning) and centrifuged at 1500 rpm for 5 min at room temperature. Cells were resuspended in 2% of heat-inactivated FBS in PBS. Cell suspensions were blocked for 5 min at 4 °C with rat anti-mouse CD16/CD32 (#553142, Mouse BD Fc Block, BD Biosciences) in FACS buffer, and incubated for 30 min with a mix of four APC-conjugated antibodies binding CD45 (#103112, Biolegend), CD31 (#102410, Biolegend), CD11b (#101212, Biolegend), F4/80 (#123116, BioLegend), CD19 (#115512, Biolegend), and TER-119 (#116212, Biolegend). 300 nM DAPI was added as a live-cell marker. Individual cancer cell suspensions were incubated for 30 min with hashtag oligonucleotide-conjugated antibodies in addition to FACS antibodies.
Sorted cell suspensions were prepared for scRNA-seq using the 3′ v3 10X Genomics Chromium platform according to manufacturer’s instructions. Briefly, sorted cells were washed once with PBS containing 1% bovine serum albumin (BSA) and resuspended in PBS containing 1% BSA to a final concentration of 700–1,300 cells per μl. The viability of cells in all experiments was above 80%, as confirmed with 0.2% (w/v) Trypan Blue staining (Countess II, Invitrogen). Cells were captured in droplets. Following reverse transcription and cell barcoding in droplets, emulsions were broken, and cDNA purified using Dynabeads MyOne SILANE followed by PCR amplification as per manufacturer’s instructions. Between 20,000 to 25,000 cells were targeted for each droplet formulation lane. Samples were multiplexed together in the lanes following the TotalSeq B cell hashing protocol. Final libraries were sequenced on Illumina NovaSeq S4 platform (R1 – 28 cycles, i7 – 8 cycles, R2 – 90 cycles).

value: subcutanous tumors

value: KP tumor cells

value: primary lung tumor cells


title: SS2iTregORAIinh_1


title: shKras,dox,7days,HTO

source_name: lung

value: lung

value: primary lung epithelial cells


title: Donor 9429 endogenous GIRK2, alcohol-naïve, batch a, bio rep 3


title: Donor 9429 endogenous GIRK2, alcohol-naïve, batch b, bio rep 1


title: Batch1_Patient11600-15149_BG02_1

description:
BG02_1Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: Donor 9429 endogenous GIRK2, alcohol-naïve, batch a, bio rep 2


title: Batch1_Patient11600-15149_BG01_2

description:
BG01_2Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: Batch1_Patient11600-15149_BF12_2

description:
BF12_2Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: KP,mrtx,20days,HTO


title: Batch1_Patient11600-15149_BG01_1

description:
BG01_1Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: Donor 9429 endogenous GIRK2, alcohol-naïve, batch a, bio rep 1


title: Batch1_Patient11600-15149_BF12_1

description:
BF12_1Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: shKras,dox,20days,HTO


title: Batch1_Patient11600-15149_BF11_2

description:
BF11_2Aligned.sortedByCoord.out.bam
counts_batch1.csv


title: SS2iTregORAIinh_2


title: KP,mrtx,5days,HTO


title: Donor 9429 endogenous GIRK2, alcohol-chronic, batch b, bio rep 2


title: Donor 9429 endogenous GIRK2, alcohol-chronic, batch b, bio rep 1


title: shKras,dox,5days,HTO


title: Donor 9429 endogenous GIRK2, alcohol-chronic, batch b, bio rep 3


title: subq,MRTX,BFPneg,HTO