title: HCT116, KO1, REP2

data_processing:
Clustering and sequencing  The clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumia) according to the manufacturer’s instructions. After cluster generation, the library preparations were sequenced on an Illumina Novaseq6000 platform and 150 bp paired-end reads were generated.
Data Analysis   Quality control 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. At the same time, Q20, Q30 and GC content the clean data were calculated. All the downstream analyses were based on the clean data with high quality.
Reads mapping to the reference genome  STAR is used to align clean reads to reference genome. STAR outperforms other aligners by a factor of >50 in mapping speed, while at the same time improving alignment sensitivity and precision. In addition to unbiased de novo detection of canonical junctions, STAR can discover non-canonical splices and chimeric (fusion) transcripts, and is also capable of mapping full-length RNA sequences. [Dobin A1, Davis CA, Schlesinger F, Drenkow J, et.al. STAR: ultrafast universal RNA-seq aligner.Bioinformatics. 2013 Jan;29(1):15-21. doi: 10.1093/bioinformatics/bts635. Epub 2012 Oct 25.]
Quantification of gene expression level  HTSeq v0.6.0 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. FPKM, expected number of Fragments Per Kilobase of transcript sequence per Millions base pairs sequenced, considers the effect of sequencing depth and gene length for the reads count at the same time, and is currently the most commonly used method for estimating gene expression levels.
Differential expression analysis We applied DESeq2 algorithm to filter the differentially expressed genes, after the significant analysis and FDR analysis under the following criteria: i) |log2FC| > 1 ; ii) Pvalue < 0.05.
Assembly: hg18
Supplementary files format and content: tab-delimited text files include RPKM values for each Sample


title: P1-TI-scRNAseq

status: Public on Apr 23 2022

type: SRA

city: Dalian

first: Hai-long

last: Piao

state: Liaoning

address: No.457 Zhongshan Road

country: China

institute: Dalian Institute of chemical physics

description: 10x Genomics

library_source: transcriptomic single cell

data_processing:
Raw fastq files were quantified to unique molecular identifiers (UMI) count values with Cell Ranger toolkit (version 4.0.0) with default parameters. The filtered feature-barcode matrix was utilized.
Supplementary files format and content: Tab-separated  matrix files, row names represent features and column names represent barcodes

molecule: total RNA

organism: Homo sapiens

source_name: Lung

extract_protocol:
The resected tumor samples were washed by phosphate buffered saline (PBS) and completely immersed in MASC tissue storage solution (Miltenyi, Germany) at 4 ℃.Tumor dissociation for 10X Single Cell RNA Sequencing according to protocol (CG000147, https://assets.ctfassets.net/an68im79xiti/2s0LZ3yI8OMEheRtFo9Szc/1fbfbd97ec3e574b6ab8b8fa12eef0cd/CG000147_Demonstrated_Protocol_Tumor_Dissociation_RevB.pdf). Using a wide-bore pipette tip, gently and thoroughly mix the cell suspensions. Determine the cell concentration using a Countess® II Automated Cell Counter. Centrifuge cells at 300 rcf for 5 min. Remove supernatant without disrupting the cell pellet. Using a wide-bore pipette tip, add 1 ml 1X PBS with 0.04% BSA to the tube. Gently pipette mix 5 times and invert tubes to resuspend the cell pellet. 10x Genomics (US) Single Cell Protocols (CG000053). Cell Counting & Quality Control. Thoroughly vortex 0.4% trypan blue stain and centrifuge briefly. Using a wide-bore pipette tip, gently and thoroughly mix the cells. Immediately, aliquot 10 µl cells and mix with 10 µl 0.4% trypan blue stain. Gently pipette mix. Transfer 10 µl trypan blue stained cells to a chamber on Countess® II Cell Counting Slide and determine the cell concentration and cell viability.
The single cell suspension was loaded into Chromium microfluidic chips with 3’ v2 chemistry and barcoded with a 10× Chromium Controller (10X Genomics, US). RNA from the barcoded cells was subsequently reverse-transcribed and sequencing libraries constructed with reagents from a Chromium Single Cell 3’ v2 reagent kit (10X Genomics, US) according to the manufacturer’s instructions (CG000108). The Chromium™ Single Cell 3’ v2 Protocol (CG00052) produces Single Cell 3’ libraries, ready for Illumina Novaseq PE150 platform. Briefly, GCs were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was retrotranscripted to cDNA, which contains an Ilumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the apropiated sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.

tag: tissue

value: Lung

tag: cell type

value: Human lung

tag: genome build


title: Root, F142, high P, rep1

city: Montpellier

first: Christophe

last: Maurel

address: 2 place Pierre Viala

department: Water, signaling and hydraulic architecture

country: France

institute: Institute for Plant Sciences of Montpellier (IPSiM)

library_source: transcriptomic

data_processing:
Sequence reads were trimmed for adaptor sequence/low-quality sequence using fastp tool  (parameter: --qualified_quality_phred 20 --unqualified_percent_limit 50 –n_base_limit 10)
Trimmed sequence reads were mapped to B73_RefGen_v4, release 44 using hisat2
Read count extraction were performed using htseq-count (parameter: --mode union)
Assembly: Zea_mays.B73_RefGen_v4, release 44
Supplementary files format and content: Tab-separated text files include read count values for each sample
Supplementary files format and content: Tab-separated text files include FPKM values for each sample

organism: Zea mays

source_name: Root

extract_protocol:
RNA was harvested using innuPREP Plant RNA Kit (Analytik Jena AG, Jena, Germany).
RNA libraries for RNA-seq were prepared using NEBNext® UltraTM RNA Library Prep Kit following manufacturer's protocols.

value: Root

tag: genotype

tag: treatment

value: high P


title: Root, F142, high P, rep2


title: Root, SF1, high P, rep1


title: P1-TM-scRNAseq

description: 10x Genomics

data_processing:
Raw fastq files were quantified to unique molecular identifiers (UMI) count values with Cell Ranger toolkit (version 4.0.2) with default parameters. The filtered feature-barcode matrix was utilized.
Supplementary files format and content: Tab-separated  matrix files, row names represent features and column names represent barcodes


title: P2-NM-scRNAseq

description: 10x Genomics

data_processing:
Raw fastq files were quantified to unique molecular identifiers (UMI) count values with Cell Ranger toolkit (version 4.0.7) with default parameters. The filtered feature-barcode matrix was utilized.
Supplementary files format and content: Tab-separated  matrix files, row names represent features and column names represent barcodes


title: C6-5_DAL2921

city: Egham

first: Gerhard

last: Leubner-Metzger

state: Surrey

address: Bourne Building 3-30

department: Biological Sciences

country: United Kingdom

institute: Royal Holloway University of London

data_processing:
Unitigs were assembled in ABySS
collapsed into a single sequence set using CD-HIT-EST at 98% identity
Collapsed unitigs were assemble using MIRA and all contigs combined using ABySS
Only those transcripts with a minimum length of 200 bp were retained
Reads were mapped to the final assembly using BWA, counts generated with Htseq
Supplementary files format and content: tab delimited data matrix of counts for each sample

molecule: polyA RNA

organism: Lepidium sativum

source_name: seed

growth_protocol: Seeds were imbibed in treatments indicated

extract_protocol:
Messenger RNA was enriched by polyA isolation using an NEBNext® Poly(A) mRNA Magnetic Isolation Module (New England Biolabs (NEB), Ipswich, MA  , USA)
Libraries were prepared using NEBNext® Ultra™ II Directional RNA Library Prep Kit (NEB)

value: seed

value: L. FR14; Keimsprossen; Juliwa

value: control imbibition 6h


title: DS-1_DAL2921

treatment_protocol: Seeds were imbibed in treatments indicated

value: dry seed


title: P3-NI-scRNAseq

description: 10x Genomics

data_processing:
Raw fastq files were quantified to unique molecular identifiers (UMI) count values with Cell Ranger toolkit (version 4.0.10) with default parameters. The filtered feature-barcode matrix was utilized.
Supplementary files format and content: Tab-separated  matrix files, row names represent features and column names represent barcodes


title: C6-4_DAL2921


title: P3-NI-ST

description: 10x Genomics

data_processing:
Raw fastq files  were processed by the Space Ranger (version 1.2.0). The filtered gene-spots matrix  were used for down-streaming data analysis
Assembly: GRCh38
Supplementary files format and content: Tab-separated values files and matrix files
Library strategy: spatial transcriptomics

source_name: normal

extract_protocol:
The resected tissues were trimmed to cubes with diameter of 0.6-1.0 cm. Use sterile filter paper to suck out blood and liquid. The tissue was completely immersed in OCT and placed in liquid nitrogen for cryoembedding and stored in – 80℃.  Then, prepare 10 frozen sections (10 um) for every sample and extract RNA with Qiagen kit for 2100 detection. Samples with RIN>7 were eligible. The Visium Spatial Tissue Optimization Slide & Reagent kit (10X Genomics, US) was used to optimize permeabilization conditions for the tissue according to Visium Spatial Tissue Optimization User Guide (CG000238, https://support.10xgenomics.com/). Briefly, the Visium Spatial Tissue Optimization workflow includes placing tissue sections on 7 Capture Areas on a Visium Tissue Optimization slide. The sections are fixed, stained, and then permeabilized for different times. mRNA released during permeabilization binds to oligonucleotides on the Capture Areas. Fluorescent cDNA is synthesized on the slide and imaged. The permeabilization time that results in maximum fluorescence signal with the lowest signal diffusion is optimal. If the signal is the same at two time points, the longer permeabilization time is considered optimal.
Visium Spatial Gene Expression Reagent Kits User Guide (CG000239) was used to generate Visium Spatial Gene Expression libraries. 4 capture areas, each with ~5000 unique gene expression spots, were included. Each capture area is 6.5 x 6.5 mm and is surrounded by a fiducial frame for a total area of 8 x 8 mm. A readable label with a serial number defines the active surface of the slide. Tissue sections are always placed on the capture areas on the active surface.Sequencing was performed with a Novaseq 6000 PE150 platform according to the manufacturer’s instructions (Illumina, US) at an average depth of 300 million read-pairs per sample. Enzymatic fragmentation and size selection are used to optimize the cDNA amplicon size. P5, P7, i7 and i5 sample indexes, and TruSeq Read  2 (read 2 primer sequence) are added via End Repair, A-tailing, Adaptor Ligation, and PCR. The final libraries contain the P5 and P7 primers used in Illumina amplification. A Visium Spatial Gene Expression library comprises standard Illumina paired-end constructs which begin and end with P5 and P7. The 16 bp Spatial Barcode and 12 bp UMI are encoded in Read 1, while Read 2 is used to sequence the cDNA fragment. i7 and i5 sample index sequences are incorporated. TruSeq Read 1 and TruSeq Read 2 are standard Illumina sequencing primer sites used in paired-end sequencing.

tag: disease state

value: lung cancer

value: normal


title: Root, F160, high P, rep1


title: P3-TM-ST

description: 10x Genomics

source_name: tumor

value: tumor


title: P3-TI-ST

description: 10x Genomics


title: Ctx glia-N+F

city: Seoul

first: Yunseon

last: Yang

address: 222, Wangsimni-ro

country: South Korea

institute: Hanyang University

data_processing:
Illumina Casava1.8 software used for basecalling/1000000019358 v02
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence(using fastx_trimmer), then mapped to hg19 whole genome using Tophat/HISAT2
Read count extraction and nomalization were performed using edgeR/quantile normalization
Assembly: hg19
Supplementary files format and content: tab-delimited text files include read count values for each Sample

organism: Mus musculus

source_name: Cortical glia of postnatal day 1 ICR mouse pup

extract_protocol:
RNA was harvested using Trizol reagent (Invitrogen). 1.3 ug of total RNA was used for the construction of sequencing libraries.
RNA libraries were prepared for sequencing using SMARTER mRNA-Seq Library Prep Kit standard protocols.

value: Cortical glia

tag: gene manipulation

value: Nurr1+Foxa2 (Lentivirus)

value: Amyloid beta


title: Root, F160, high P, rep2


title: C6-1_DAL2921


title: P2-NM-ST

description: 10x Genomics


title: C6-3_DAL2921


title: P2-NI-ST

description: 10x Genomics


title: Ctx glia-Cont

value: Control (Lentivirus)


title: P3-NM-ST

description: 10x Genomics


title: Root, F160, high P, rep3


title: Root, F2, high P, rep2


title: Root, F2, high P, rep3


title: C12-5_DAL2921

value: control imbibition 12h


title: Root, F2, high P, rep1


title: Root, EP1, high P, rep3


title: P2-TI-ST

description: 10x Genomics


title: Hippocampal glia-Cont

source_name: Hippocampal glia of postnatal day 1 ICR mouse pup

value: Hippocampal glia


title: C6-2_DAL2921


title: P2-TM-ST

description: 10x Genomics

value: Lung


title: C12-3_DAL2921


title: C12-4_DAL2921


title: P1-TM-ST

description: 10x Genomics

value: Human lung


title: Hippocampal glia-N+F


title: P1-NM-ST

description: 10x Genomics


title: C12-2_DAL2921


title: Root, EP1, high P, rep2

value: Root


title: Hippocampal glia


title: P1-NI-ST

description: 10x Genomics


title: Root, EP1, high P, rep1

value: high P


title: C12-1_DAL2921

value: seed


title: P1-TI-ST

description: 10x Genomics


title: Ctx glia


title: RNA SCO7099_dC_3

status: Public on Apr 22 2022

city: Berkeley

first: Hiroshi

last: Otani

state: CA

address: 1 Cyclotron Road

department: DOE Joint Genome Institute

country: USA

institute: Lawerence Berkeley National Laboratory

data_processing:
Raw reads were scanned from 3’ to 5’ and those with a quality score value below 20 were trimmed and reads consisting of fewer than 35 nucleotides were discarded using BBDuk (sourceforge.net/projects/bbmap/)
Trimmed reads were aligned to the S. coelicolor A3(2) chromosome and ERCC92 sequences using HISAT2 with the “no-spliced-alignment” option and the “maxins” option of 1,000
The number of fragments overlapping each gene was counted using featureCounts
Fragment counts were normalized by ERCC fragment counts using the R package RUVSeq and differential expression was analyzed using the R package DESeq2
Genome_build: Streptomyces coelicolor A3(2)
Supplementary_files_format_and_content: Fragment counts for each feature.

organism: Streptomyces coelicolor A3(2)

source_name: In vitro transcription

growth_protocol: The genomic DNA of S. coelicolor A3(2) was isolated by using GenElute Bacterial Genomic DNA kit (MilliporeSigma) and digested by EcoRI, HindIII, BamHI or XhoI. Equal quantities of the genomic DNA solutions treated by different restriction enzymes were combined. In vitro transcription assays were performed using Echo® 525 LIQUID HANDLER (Labcyte). Two µl of 8 µM sigma factor, 1 µl of 1 unit/µl E. coli RNA polymerase core enzyme (New England BioLabs) and 2 µl of 5X E. coli RNA polymerase reaction buffer were mixed and incubated at 30ºC for 15 min. To this mixture, 1 µl of 200 ng/µl digested genomic DNA mixture was added and the mixture was incubated at 30ºC for 15 min. The in vitro transcription reaction was initiated by adding 2 µl of 2.5 mM NTP mixture (Invitrogen) and the mixture was incubated at 30ºC for 20 min. To the reaction was the DNase solution consisting of 2 µl TURBO DNase (2 units/µl; Ambion), 1.5 µl of 10X TURBO DNase buffer and 1 µl of ERCC RNA Spike-in Mix (Invitrogen) diluted by 100 times added. The reaction was incubated at 37ºC for 30 min and the RNAs were purified using RNeasy kit (Qiagen).

extract_protocol:
RNeasy kit (Qiagen)
TruSeq Stranded RNA Library Prep Kit (Illumina)

treatment_protocol: With or without a sigma factor

tag: sample type

value: Sigma factor SCO7099_dC, replicate # 3


title: RNA SCO7099_dC_2

value: Sigma factor SCO7099_dC, replicate # 2


title: RNA SCO7099_dC_4

value: Sigma factor SCO7099_dC, replicate # 4


title: RNA SCO6520_4

value: Sigma factor SCO6520, replicate # 4


title: RNA SCO7099_dC_1

value: Sigma factor SCO7099_dC, replicate # 1


title: RNA SCO6520_3

value: Sigma factor SCO6520, replicate # 3


title: RNA SCO6520_1

value: Sigma factor SCO6520, replicate # 1


title: RNA SCO6520_2

value: Sigma factor SCO6520, replicate # 2


title: RNA SCO6239_4

value: Sigma factor SCO6239, replicate # 4


title: RNA SCO6239_3

value: Sigma factor SCO6239, replicate # 3


title: RNA SCO6239_1

value: Sigma factor SCO6239, replicate # 1


title: OCH053_tumor

type: RNA

city: Sendai

first: Yasufumi

last: Otsuki

address: 4-1 Seiryo-machi, Aobaku

country: Japan

institute: Tohoku University

hyb_protocol: Hybridization was performed according to the Clariom D standard protocol

scan_protocol: Scanning was performed according to the Clariom D standard protocol

data_processing: The data quality of *.CEL sample files was analyzed by Transcriptome Analysis Console 4.0. Data was normalized by Signal Space Transformation-Robust Multiarray Analysis (sst-RMA) method.

label: biotin

source_name: formalin-fixed and paraffin embedded primary tumor samples

label_protocol: Labelling was performed according to  the Clariom D standard protocol

extract_protocol: The RNA was extracted from tissues fixed in formaldehyde, paraffin-embedded (FFPE) and stored at room temperature. Four 5-μm sections or two 10-μm sections from FFPE of tumor tissue and normal colonic mucosa were used. Extraction of RNA material was performed using the commercially available kit RNeasy FFPE Kit (QIAGEN, Hilden, Nordrhein-Westfalen, Germany).

value: colorectal cancer


title: OCH055_tumor


title: RNA SCO5820_dN_1

value: Sigma factor SCO5820_dN, replicate # 1


title: RNA SCO5820_dN_4

value: Sigma factor SCO5820_dN, replicate # 4


title: RNA SCO5820_dN_3

value: Sigma factor SCO5820_dN, replicate # 3


title: RNA SCO5820_3

value: Sigma factor SCO5820, replicate # 3


title: RNA SCO5820_dN_2

value: Sigma factor SCO5820_dN, replicate # 2


title: OCH044_tumor


title: OCH019_normal

source_name: formalin-fixed and paraffin embedded normal colorectal mucosa samples

value: normal colorectal mucosa


title: RNA SCO5820_4

value: Sigma factor SCO5820, replicate # 4


title: OCH053_normal


title: RNA SCO5820_1

value: Sigma factor SCO5820, replicate # 1


title: OCH051_tumor


title: OCH047_normal


title: OCH045_tumor


title: OCH007_normal


title: OCH055_normal


title: OCH044_normal


title: OCH043_normal


title: OCH047_tumor


title: OCH051_normal


title: OCH007_tumor


title: OCH043_tumor


title: RNA SCO5243_dN_4

value: Sigma factor SCO5243_dN, replicate # 4


title: OCH019_tumor


title: RNA SCO5243_dN_1

value: Sigma factor SCO5243_dN, replicate # 1


title: RNA SCO5243_4

value: Sigma factor SCO5243, replicate # 4


title: RNA SCO5216_4

value: Sigma factor SCO5216, replicate # 4


title: RNA SCO5243_2

value: Sigma factor SCO5243, replicate # 2


title: RNA SCO5243_3

value: Sigma factor SCO5243, replicate # 3


title: RNA SCO5243_1

value: Sigma factor SCO5243, replicate # 1


title: RNA SCO5216_3

value: Sigma factor SCO5216, replicate # 3


title: P2-TM-scRNAseq

description: 10x Genomics

data_processing:
Raw fastq files were quantified to unique molecular identifiers (UMI) count values with Cell Ranger toolkit (version 4.0.6) with default parameters. The filtered feature-barcode matrix was utilized.
Supplementary files format and content: Tab-separated  matrix files, row names represent features and column names represent barcodes

value: Lung

value: Human lung


title: RNA SCO4895_2

value: Sigma factor SCO4895, replicate # 2


title: RNA SCO5216_2

value: Sigma factor SCO5216, replicate # 2


title: RNA SCO5216_1

value: Sigma factor SCO5216, replicate # 1


title: RNA SCO4895_1

value: Sigma factor SCO4895, replicate # 1


title: OCH045_normal

value: normal colorectal mucosa


title: RNA SCO4895_4

value: Sigma factor SCO4895, replicate # 4


title: RNA SCO4769_4

value: Sigma factor SCO4769, replicate # 4


title: RNA SCO4769_3

value: Sigma factor SCO4769, replicate # 3


title: RNA SCO4895_3

value: Sigma factor SCO4895, replicate # 3


title: RNA SCO4769_2

value: Sigma factor SCO4769, replicate # 2


title: RNA SCO4769_1

value: Sigma factor SCO4769, replicate # 1


title: RNA SCO4035_2

value: Sigma factor SCO4035, replicate # 2


title: RNA SCO4035_4

value: Sigma factor SCO4035, replicate # 4


title: RNA SCO4035_1

value: Sigma factor SCO4035, replicate # 1


title: RNA SCO3626_3

value: Sigma factor SCO3626, replicate # 3


title: RNA SCO3626_4

value: Sigma factor SCO3626, replicate # 4


title: RNA SCO3068_4

value: Sigma factor SCO3068, replicate # 4


title: RNA SCO3626_1

value: Sigma factor SCO3626, replicate # 1


title: RNA SCO3068_1

value: Sigma factor SCO3068, replicate # 1


title: RNA SCO2742_4

value: Sigma factor SCO2742, replicate # 4


title: RNA SCO2742_3

value: Sigma factor SCO2742, replicate # 3


title: RNA SCO0864_2

value: Sigma factor SCO0864, replicate # 2


title: RNA SCO2742_dC_3

value: Sigma factor SCO2742_dC, replicate # 3


title: RNA SCO2742_dC_2

value: Sigma factor SCO2742_dC, replicate # 2


title: RNA SCO0864_3

value: Sigma factor SCO0864, replicate # 3


title: RNA SCO0864_4

value: Sigma factor SCO0864, replicate # 4


title: RNA SCO2742_1

value: Sigma factor SCO2742, replicate # 1


title: RNA SCO2742_2

value: Sigma factor SCO2742, replicate # 2


title: RNA SCO4035_3

value: Sigma factor SCO4035, replicate # 3


title: RNA SCO3068_3

value: Sigma factor SCO3068, replicate # 3


title: RNA SCO0864_1

value: Sigma factor SCO0864, replicate # 1


title: TSS SCO7099_dC_4

data_processing:
Adaptor sequences present at the 3’ end of reads were trimmed using BBDuk
Raw reads were scanned from 3’ to 5’ and those with a quality score value below 20 were trimmed and reads consisting of fewer than 35 nucleotides were discarded using BBDuk
Trimmed reads were aligned to the S. coelicolor A3(2) chromosome and ERCC92 sequences using HISAT2 with the “no-spliced-alignment” option and the “maxins” option of 1,000
The 5’ end of the forward reads that aligned each genomic position was counted using Samtools
5’ ends within 100 bp were clustered together. If multiple 5’ ends located nearby had standard deviation of < 10, they were subclustered together and the 5’ end that had the largest read count within the subcluster was considered the TSS. The sum of the read counts of all the 5’ ends within the subcluster was used as the read count of the TSS. ERCC92 transcripts that had at least 10 reads aligning the 1st nucleotide in all the replicates were used as normalisation control. The read count relative to the maximum number among samples were calculated for each ERCC92 transcript and the mean value of all the relative read counts were used as the normalisation factor, which ranges between 0 and 1. TSSs with the normalised read counts (read counts divided by the mean normalisation factor) of < 4 in at least one of the replicates in the sample with a sigma factor were removed from the further analysis.
Genome_build: Streptomyces coelicolor A3(2)
Supplementary_files_format_and_content: CSV file with 5' read count

extract_protocol:
RNeasy kit (Qiagen)
The triphosphates at the 5’ end of 6 µg RNA samples were converted to monophosphates by using 3.75 units of RNA 5’ pyrophosphohydrolase (RppH; New England BioLabs) at 30ºC for 1 hour. The 5’ ends of the transcripts were ligated by the adaptor (GUUCAGAGUUCUACAGUCCGACGAUC) using 15 units of T4 RNA ligase 1 (ssRNA ligase; New England BioLabs) at 25ºC for 2 hours followed by incubation at 17ºC for 18 hours. Ligated RNA samples were purified by using RNeasy Kits and RNeasy MinElute Cleanup Kits (QIAGEN). cDNA was synthesised by using a random hexamer with an adaptor sequence (GCCTTGGCACCCGAGAATTCCANNNNNN) and SuperScript IV First-Strand Synthesis System (Invitrogen). The cDNA was amplified by using KAPA Library Amplification Kit (Roche) and index primers from TruSeq Small RNA Library Preparation Kit (Illumina). Libraries were purified by using the equal volume of AMPure XP (Beckman Coulter) twice.

value: Sigma factor SCO7099_dC, replicate # 4


title: RNA SCO3626_2

value: Sigma factor SCO3626, replicate # 2


title: RNA SCO2742_dC_4

value: Sigma factor SCO2742_dC, replicate # 4


title: RNA SCO3068_2

value: Sigma factor SCO3068, replicate # 2


title: RNA No_sigma_factor_2

value: No sigma factor, replicate # 2


title: RNA No_sigma_factor_1

value: No sigma factor, replicate # 1


title: RNA SCO2742_dC_1

value: Sigma factor SCO2742_dC, replicate # 1


title: RNA No_sigma_factor_3

value: No sigma factor, replicate # 3


title: TSS SCO7099_dC_3

value: Sigma factor SCO7099_dC, replicate # 3


title: TSS SCO7099_dC_2

value: Sigma factor SCO7099_dC, replicate # 2


title: TSS SCO6520_4

value: Sigma factor SCO6520, replicate # 4


title: TSS SCO6239_4

value: Sigma factor SCO6239, replicate # 4


title: RNA No_sigma_factor_4

value: No sigma factor, replicate # 4


title: TSS SCO6520_3

value: Sigma factor SCO6520, replicate # 3


title: TSS SCO6520_2

value: Sigma factor SCO6520, replicate # 2


title: TSS SCO6239_3

value: Sigma factor SCO6239, replicate # 3


title: TSS SCO5820_dN_4

value: Sigma factor SCO5820_dN, replicate # 4


title: TSS SCO6239_2

value: Sigma factor SCO6239, replicate # 2


title: TSS SCO5820_dN_2

value: Sigma factor SCO5820_dN, replicate # 2


title: TSS SCO5820_dN_3

value: Sigma factor SCO5820_dN, replicate # 3


title: TSS SCO5820_4

value: Sigma factor SCO5820, replicate # 4


title: TSS SCO5820_3

value: Sigma factor SCO5820, replicate # 3


title: TSS SCO5243_dN_4

value: Sigma factor SCO5243_dN, replicate # 4


title: TSS SCO5243_dN_3

value: Sigma factor SCO5243_dN, replicate # 3


title: Root, F142, high P, rep3

value: Root

value: high P


title: TSS SCO5243_dN_2

value: Sigma factor SCO5243_dN, replicate # 2


title: TSS SCO5243_3

value: Sigma factor SCO5243, replicate # 3


title: TSS SCO5216_4

value: Sigma factor SCO5216, replicate # 4


title: TSS SCO5243_4

value: Sigma factor SCO5243, replicate # 4


title: TSS SCO5216_3

value: Sigma factor SCO5216, replicate # 3


title: TSS SCO5243_2

value: Sigma factor SCO5243, replicate # 2


title: TSS SCO5216_2

value: Sigma factor SCO5216, replicate # 2


title: TSS SCO4895_3

value: Sigma factor SCO4895, replicate # 3


title: TSS SCO4769_4

value: Sigma factor SCO4769, replicate # 4


title: TSS SCO4895_4

value: Sigma factor SCO4895, replicate # 4


title: TSS SCO4895_2

value: Sigma factor SCO4895, replicate # 2


title: TSS SCO4769_3

value: Sigma factor SCO4769, replicate # 3


title: TSS SCO4035_3

value: Sigma factor SCO4035, replicate # 3


title: TSS SCO4769_2

value: Sigma factor SCO4769, replicate # 2


title: TSS SCO5820_2

value: Sigma factor SCO5820, replicate # 2


title: RNA SCO6239_2

value: Sigma factor SCO6239, replicate # 2


title: RNA SCO5820_2


title: TSS SCO3068_4

value: Sigma factor SCO3068, replicate # 4


title: TSS SCO3068_3

value: Sigma factor SCO3068, replicate # 3


title: TSS SCO3626_3

value: Sigma factor SCO3626, replicate # 3


title: TSS SCO2742_dC_3

value: Sigma factor SCO2742_dC, replicate # 3


title: TSS SCO2742_dC_4

value: Sigma factor SCO2742_dC, replicate # 4


title: RNA SCO5243_dN_3

value: Sigma factor SCO5243_dN, replicate # 3


title: TSS SCO3068_2

value: Sigma factor SCO3068, replicate # 2


title: TSS SCO2742_dC_2

value: Sigma factor SCO2742_dC, replicate # 2


title: TSS SCO2742_4

value: Sigma factor SCO2742, replicate # 4


title: TSS SCO2742_3

value: Sigma factor SCO2742, replicate # 3


title: TSS SCO3626_4

value: Sigma factor SCO3626, replicate # 4


title: TSS SCO4035_4

value: Sigma factor SCO4035, replicate # 4


title: TSS No_sigma_factor_4

value: No sigma factor, replicate # 4


title: TSS SCO3626_2

value: Sigma factor SCO3626, replicate # 2


title: TSS SCO0864_2

value: Sigma factor SCO0864, replicate # 2


title: TSS SCO0864_3

value: Sigma factor SCO0864, replicate # 3


title: 4C_KvDMR1_DC_#2_technical_replicate_2

city: Columbia

first: Rocío

middle: Melissa

last: Rivera

state: MO

address: 920 East Campus Drive

department: Division of Animal Sciences

institute: University of Missouri, Columbia

description: Demultiplexed based on secondary barcode (bait sequence)

library_source: genomic

data_processing:
Library strategy: 4C-Seq
Raw sequencing reads were first sorted into the three 4C assays (KvDMR1, IGF2R_MseI, and IGF2R_BsrI) based on the secondary barcodes. This was accomplished by aligning the bait part of the reads to the bait sequences using bowtie2 (version 2.3.5.1) and filtering for unique alignments. Meanwhile, parental alleles were assigned to each read pairs based on the SNPs in the bait sequences. Then the bait sequences were removed from the reads and reads were trimmed for adapter sequences and low quality bases using trimmomatic (version 0.39) with parameters ‘ILLUMINACLIP:adapter_seq:2:30:10:1:true LEADING:20 TRAILING:20 AVGQUAL:20 MAXINFO:0:0.5’. The trimmed reads were split into multiple fragments by the recognition sites of corresponding first and second RE used. The first fragment on the 5’ side of each read was kept and read pairs were combined and aligned to genome as single-end reads using bowtie2 with parameters ‘-N 1 -L 15 --no-unal’. Aligned reads with mapping quality less than 20 were filtered out using samtools (version 1.13).
The genome was fragmented by NlaIII recognition site into NlaIII restriction fragments. Read coverage was calculated for NlaIII restriction fragments using bedtools (version 2.30.0) with parameters ‘coverage -sorted -counts’. Self-ligation reads aligned to the NlaIII restriction fragments covering the bait region and one up/downstream fragment were excluded for statistical analyses. 4C peaks were called using fourSig (version 2.2) with parameters ‘cis.only=FALSE, window.size=25, iterations=1000, fdr=0.01, fdr.prob=0.05, only.mappable=FALSE’. fourSig reported 4C peaks for individual samples were converted to NlaIII restriction fragments, and the fragments present in at least two samples in a treatment group were reported as the 4C peaks for the group. For statistical comparison between treatment groups by DESeq2 (version 1.28.1), the sum read coverage of a running window of 25 NlaIII restriction fragments were calculated and used as input. A prefilter for low coverage window using ‘rowSums(counts>2) >= 2’ of edgeR (version 3.30.3) were conducted to make the input list manageable. Default settings of DESeq2 were used for other steps. False discovery rate was controlled at 0.05 for DESeq2 results (labelled as Padj) by the Benjamini-Hochberg method. The coordinates of the 13th NlaIII restriction fragment in each running window were used to indicate that window.
Genome_build: ARS-UCD1.2
Supplementary_files_format_and_content: Read coverage for NlaIII genome fragments in BEDGRAPH format.

molecule: genomic DNA

organism: Bos indicus x Bos taurus

source_name: Day 105 fetal skin fibroblast primary cell

growth_protocol: Day 105 Bos taurus indicus (B. t. indicus; Brahman breed) x Bos taurus taurus (B. t. taurus; Angus breed) F1 hybrid fetuses were generated by our laboratory following the protocol previous described (PMID: 23751783) and used as tissue donors. Fetal skins were collected to establish fibroblast primary cell line using protocol adapted from a publication (PMID: 24198123). Fibroblast cells were cultured in bovine embryonic fibroblast medium (89% (v/v) DMEM (Gibco, 11885084), 10% (v/v) fetal bovine serum (Atlanta Biologicalsa, S11150H), and 1% (v/v) Antibiotic-antimycotic (Gibco, 15240062)) at 38.5°C with 5% CO2.

extract_protocol:
None
The 4C library preparation procedure mainly followed the Krijger protocol (PMID: 31351925) with some adaptations from other published protocols (PMID: 21938629, 22609568, and 22929766). Briefly, for each sample, six million fibroblast cells were fixed with 2% (v/v) formaldehyde at a concentration of two million cells per ml. Fixed cells were lysed, washed, and underwent first restriction enzyme (RE, NlaIII for all assays) digestion overnight. On day two, the first RE was inactivated and samples were diluted and ligated with T4 DNA Ligase overnight. On day three, samples were treated with Proteinase K overnight to reverse cross-links. On day four, samples were treated with RNase A and ligated DNA was extracted using Phenol-Chloroform. Ligated DNA samples were digested with second RE (Tsp45I, MseI, and BsrI for KvDMR1, IGF2R_MseI, and IGF2R_BsrI assays, respectively) overnight. On day five, the second RE was inactivated and DNA samples were ligated with T4 DNA Ligase overnight. On day six, DNA samples were ethanol precipitated and purified, resulting in 4C templates. 4C templates were PCR amplified with assay-specific primers and high fidelity polymerase and purified with magnetic beads for two rounds, resulting in barcoded 4C libraries. After quantification and quality check, barcoded 4C libraries were pooled for sequencing with 15% spike-in of PhiX Control to increase base diversity. For some barcodes, samples from three different 4C assays shared the same barcode and could be separated based on bait sequence (4C assay targeted region) in the reads which served as a secondary barcode. The bait sequence in the reads also contains single nucleotide polymorphisms (SNP) to differentiate parental alleles.

treatment_protocol: For bovine fetuses, artificial insemination was used to generate the control group and assisted reproductive technologies (ART) were used to generate the ART group following the protocol previous described (PMID: 23751783). The large offspring syndrome (LOS) group was defined as individuals from the ART group with body weight greater than 97th centile of controls. For fibroblast cells, to induce loss of DNA methylation, a subgroup of fibroblasts derived from control fetuses were treated with 0.5 uM Decitabine (5-aza-2'-deoxycytidine) for 96 hours (DC group). BEF medium with 0.5 uM Decitabine were changed every 24 hours to maintain the concentration of Decitabine.

tag: conception method

value: Artificial insemination

tag: developmental stage

value: D105 fetus

tag: disease status

value: Healthy

value: fibroblast

value: 0.5 uM Decitabine for 96 hours cell culture

tag: 4c bait

tag: 4c first restriction enzyme

value: NlaIII

tag: 4c second restriction enzyme


title: 4C_KvDMR1_LOS_#3_technical_replicate_3

value: Assisted reproductive technology

value: LOS


title: TSS SCO2742_2

value: Sigma factor SCO2742, replicate # 2


title: 4C_KvDMR1_DC_#4


title: 4C_KvDMR1_Control_#4_technical_replicate_3


title: 4C_KvDMR1_Control_#4_technical_replicate_2


title: 4C_KvDMR1_DC_#2_technical_replicate_3


title: 4C_KvDMR1_LOS_#3_technical_replicate_2


title: 4C_KvDMR1_Control_#2_technical_replicate_3


title: 4C_KvDMR1_Control_#2_technical_replicate_2


title: 4C_KvDMR1_DC_#3


title: TSS SCO0864_4

value: Sigma factor SCO0864, replicate # 4


title: TSS SCO4035_2

value: Sigma factor SCO4035, replicate # 2


title: 4C_KvDMR1_LOS_#3


title: 4C_KvDMR1_DC_#1


title: 4C_KvDMR1_Control_#3


title: TSS No_sigma_factor_3

value: No sigma factor, replicate # 3


title: 4C_KvDMR1_Control_#4


title: 4C_IGF2R_BsrI_DC_#4

value: IGF2R ICR

value: BsrI


title: 4C_KvDMR1_Control_#1


title: TSS No_sigma_factor_2

value: No sigma factor, replicate # 2


title: 4C_IGF2R_BsrI_DC_#3


title: 4C_IGF2R_BsrI_DC_#2


title: 4C_IGF2R_BsrI_DC_#1


title: 4C_IGF2R_BsrI_LOS_#4


title: 4C_IGF2R_BsrI_LOS_#3


title: 4C_KvDMR1_DC_#2


title: 4C_IGF2R_BsrI_LOS_#2


title: 4C_IGF2R_BsrI_LOS_#1


title: 4C_IGF2R_BsrI_Control_#1


title: 4C_IGF2R_MseI_DC_#1

value: MseI


title: 4C_IGF2R_BsrI_Control_#2


title: 4C_IGF2R_BsrI_Control_#4


title: 4C_IGF2R_MseI_LOS_#3


title: 4C_IGF2R_MseI_LOS_#1


title: 4C_IGF2R_MseI_DC_#2


title: 4C_IGF2R_MseI_DC_#3


title: 4C_KvDMR1_Control_#2


title: 4C_IGF2R_MseI_DC_#4


title: 4C_IGF2R_MseI_LOS_#2


title: 4C_IGF2R_MseI_LOS_#4


title: RNA SCO5243_dN_2

value: Sigma factor SCO5243_dN, replicate # 2


title: 4C_IGF2R_MseI_Control_#3

value: Artificial insemination


title: 4C_IGF2R_MseI_Control_#2

value: D105 fetus


title: WT H37Rv B

city: CHANDIGARH

first: Dibyendu

last: Sarkar

address: SECTOR 39A

country: India

institute: CSIR-IMTECH

description: For growth, mycobacterial strains were grown to mid log phase (OD600 0.4-0.6) and RNAs were isolated.

data_processing:
Basecalling, demultiplication and adaptor trimming was performed by Illumina HiSeq control software
Quality of raw reads was assessed by using FastQC tool
Clean reads that passed the quality filter were mapped to the reference genome (Mycobacterium tuberculosis H37Rv accession number NC_000962.3) using Hisat2.
Differential gene expression analysis of samples with respect to control was performed using Cuffdiff program (v2.2.1).
Genome_build: NC_000962.3
Supplementary_files_format_and_content: tab-delimited text files include FPKM values for each Sample ...

organism: Mycobacterium tuberculosis

source_name: WT H37Rv

growth_protocol: M. tuberculosis H37Rv strains were grown at 37°C in Middlebrook 7H9 broth (Difco) containing 0.2% glycerol, 0.05% Tween-80 and 10% Middlebrook ADC (albumin-dextrose-catalase).

extract_protocol:
RNA was extracted from exponentially growing mycobacterial cells in Middlebrook 7H9 media. Briefly, 25 ml of bacterial culture was grown to mid-log phase (OD600= 0.4 to 0.6) and combined with 40 ml of 5 M guanidinium thiocyanate solution containing 1% β-mercaptoethanol and 0.5% Tween 80. Cells were pelleted by centrifugation, and lysed by re-suspending in 1 ml Trizol (Ambion) in the presence of Lysing Matrix B (100 µm silica beads; MP Bio) using a FastPrep-24 bead beater (MP Bio) at a speed setting of 6.0 for 30 seconds. The procedure was repeated for 2-3 cycles with incubation on ice in between pulses. Next, cell lysates were centrifuged at 13000 rpm for 10 minutes; supernatant was collected and processed for RNA isolation using Direct-ZolTM RNA isolation kit (ZYMO) as per manufacturer’s recommendation. Following extraction, RNA was treated with DNAse I (Promega) to degrade contaminating DNA, and integrity was assessed using a Nanodrop (ND-1000, Spectrophotometer). RNA samples were further checked for intactness of 23S and 16S rRNA using formaldehyde-agarose gel electrophoresis, and Qubit fluoremeter (Invitrogen).
Total RNA was isolated using Direct-zol RNA MiniPrep kit (Zymo Research Corporation), the purity of isolated RNA was determined using the NanoDrop (Thermo Scientific), quantified using Qubit (Invitrogen), and RNA integrity was checked using Agilent 2200 Tape Station system (Agilent Technologies).  Library construction, RNA-sequencing and data analysis have been carried out by Agrigenome Labs Private Limited (Cochin), India. Briefly, The rRNA was removed using ‘si-tools Pan-prokaryote ribopool probes,’ and further library preparation was carried out using TruSeq Stranded RNA library prep kit. Prepared libraries were sequenced using Illumina HiSeq x10 platform, and 150-bp paired-end reads were generated. Thereads that passed quality control were mapped onto the reference genome of M. tuberculosis H37Rv using Hisat2 (version-2.0.5).  The abundance of transcripts in individual samples was estimated using Cufflinks (v2.2.1).

treatment_protocol: Cultures were grown till mid log phase (OD600 0.4-0.6), RNA was isolated and processed for sequencing.

tag: strain


title: 4C_IGF2R_MseI_Control_#4

value: Healthy


title: 4C_IGF2R_MseI_Control_#1

value: fibroblast


title: H37Rv-pde B


title: WT H37Rv A


title: phoP ko A

source_name: phoP ko


title: phoP ko B


title: CD8αβ T cells from liver

city: GuangZhou

first: bian

last: zhenhua

state: GuangDong

address: Panyu University Town

institute: South China University of Technology

data_processing:
we used MIXCR to extract the tcr repertoire from RNA sequencing
Genome_build: mm10
Supplementary_files_format_and_content: Matrix table with raw clonotype counts, types and other details for samples

source_name: CD8αβ T cells from liver

extract_protocol:
sorted CD8 T cells were removed, flash frozen on dry ice, and RNA was harvested using Trizol reagent. 1 ug of total RNA for the construction of sequencing libraries.
RNA libraries were prepared for sequencing using standard Illumina protocols

tag: age

value: 10 weeks

value: dnTGF{beta}RII

value: CD8{alpha}{beta} T cells


title: H37Rv-pde A


title: 4C_IGF2R_BsrI_Control_#3

value: NlaIII


title: 293T-DdCBE-ND5.1L_DddA.C_MTS.PD_rep1

city: Beijing

first: Hua-nan

last: Zhao

state: Beijing

address: 5 Yiheyuan Road Haidian District

department: School of Life Sciences

institute: Peking University

description:
Detect-seq
293T-DdCBE-ND5.1.FinalList.bed.gz

data_processing:
Library strategy: Detect-seq
Cut adapter step: The raw FASTQ files are firstly but the sequencing adapters with cutadapt software with the following command: "cutadapt --times 1  -e 0.1  -O 3  --quality-cutoff 25  -m 50 -a  AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC  -A  AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTAGATCTCGGTGGTCGCCGTATCATT -o {read1.out}  -p {read2.out}  {read1.in}  {read2.in}"
Alignment step: After the cut adapter step, all the FASTQ files are mapped to the reference genome, hg38 for HEK293T with bowtie2 aligner. And only record the best hit of alignment for each read.
BAM convert step:The alignment SAM results are converted to sorted BAM files with samtools software
Remove duplication step: The sorted BAM files are removed the PCR amplification bias with the PICARD MarkDuplicates command.
Off-target regions were found using Detect-seq tool(http://detect-seq.com/)
Genome_build: hg38
Supplementary_files_format_and_content: bed file

source_name: HEK293T cells

extract_protocol: Extracted genomic DNA was fragmented into ~300 bp through Covaris Focused-ultrasonicator™ Instrument (ME220), followed by a purification using DNA Clean & Concentrator-5 (Vistech, DC2005). ~5 ug DNA fragments and 10 pg spike-in model sequences were subjected to end repair with NEBNext End Repair Module (NEB, E6050), and 1 μl E. coli ligase (NEB, M0205) was added to repair nicks in DNA. Then hydroxylamine protection of endogenous 5fC was performed in 100 mM MES buffer (pH 5.0), 10 mM O-ethylhydroxylamine (EtONH2, Aldrich, 274992) at 37 °C for 6 h. The product underwent 5fC protection was diluted 2.5 times and purified with DNA Clean & Concentrator-5. Next, dA-tailing procedure was carried out using NEBNext dA-Tailing Module (NEB, E6053) followed by a DNA purification with 1.8 × AMPure XP beads.  DNA damage that may interfere the subsequent labeling step was repaired in a mixture of 2 μl endonuclease IV (Endo IV, NEB, M0304), 1 μl Bst full-length polymerase (NEB, M0328), 2 μl Taq ligase (NEB, M0208), 1 μl NAD+ (NEB, B9007), 1 μl dNTP (2.5 mM each) in NEBuffer 3 for 1 h at 37 °C and 1 h at 45 °C. DNA was purified and subjected to in vitro BER labeling reaction containing a mixture of 1 μl UDG (NEB, M0280), 1.5 μl Endo IV, 0.8 μl Bst full-length polymerase, 1.7 μl Taq ligase, 1 μl NAD+, 200 nM biotin-dUTP, 800 nM d5fCTP, 200 nM dATP and 200 nM dGTP in NEBuffer 3 for 40 min at 37 °C. The DNA was incubated with 75 mM of malononitrile in 10 mM Tris-HCl (pH 7.0) at 37°C for 20 h in an enclosed thermomixer (Eppendorf, 850 rpm), away from light. The reaction product was then purified with 1.8 × AMPure XP beads.  Labeled fragments were enriched by streptavidin C1 beads (Invitrogen) following the manufacturer's instruction. Y adaptor (see Supplementary Note 1) was ligated (NEBNext Quick Ligation Module, E6056) to double-stranded DNA on streptavidin C1 beads so that free adaptor can be removed by three-times washing of 1×B&W buffer (5 mM Tris-HCl, pH 7.5, 0.5 mM EDTA, 1 M NaCl, 0.05% Tween-20). After washing once with 1× SSC buffer (pH 7.0), beads with biotinylated DNA strands were resuspended in freshly prepared 0.15 M NaOH and incubated at room temperature for 10 min, followed by a sequential washing of 1× SSC buffer (7.0) and 1× TE buffer (pH 7.5) for once. The DNA was eluted from C1 beads using deionized water after heating at 95 °C for 3 min.   Eluted DNA was finally subjected to library amplification with indexed primers (NEB). To obtain sufficient DNA template, 2 rounds of amplification were first performed by using MightyAmp DNA polymerase (Takara), which was proved to read through regions containing biotin-dU and d5fC-malononitrile sites more easily. The PCR product was recovered via DNA Clean & Concentrator-5 and then PCR amplified for ~12 cycles using NEBNext® Q5® Hot Start HiFi PCR Master Mix (NEB, M0543). The constructed libraries were checked using the Agilent 2100 bioanalyzer. Sequencing was performed by Illumina Hiseq X Ten.

tag: cell line

value: HEK293T cells


title: 293T-DdCBE-ND6L_DddA.C_MTS.PD_rep1

description:
Detect-seq
293T-DdCBE-ND6.FinalList.bed.gz


title: CD8αα T cells from liver

source_name: CD8αα T cells from liver

value: CD8{alpha}{alpha} T cells


title: 293T-DdCBE-ND4L_DddA.C_MTS.PD_rep1

description:
Detect-seq
293T-DdCBE-ND4.FinalList.bed.gz


title: 293T-DdCBE-Lipo2000-ND6-All-PD_rep3

value: treated with ND6-L1397N


title: 293T-DdCBE-Lipo2000-ND6-All-PD_rep2


title: 293T-DdCBE-Lipo2000-ND5.1-All-PD_rep3

value: treated with ND5.1-L1397N


title: 293T-DdCBE-L1397C-ND5.3-All-PD_rep2

description:
Detect-seq
293T-DdCBE-ND5.3.FinalList.bed.gz

value: treated with ND5.3-L1397C


title: 293T-DdCBE-Lipo2000-ND4-All-PD_rep3

value: treated with ND4-L1397N


title: 293T-DdCBE-L1397C-ND5.3-All-PD_rep1


title: 293T-DdCBE-L1397C-ND4-All-PD_rep2

value: treated with ND4-L1397C


title: 293T-DdCBE-L1397C-ND4-All-PD_rep1


title: 293T-DdCBE-Dead-ND6-All-PD_rep1

value: treated with ND6-L1397N-Dead-DddA


title: 293T-DdCBE-Lipo2000-ND4-All-PD_rep2


title: 293T-DdCBE-Dead-ND5.1-All-PD_rep1

value: treated with ND5.1-L1397N-Dead-DddA


title: 293T-DdCBE-DddA.N_ND6R_MTS.PD_rep1


title: 293T-DdCBE-DddA.N_ND4R_MTS.PD_rep1


title: 293T-DdCBE-Dead-ND4-All-PD_rep1

value: treated with ND4-L1397N-Dead-DddA


title: 293T-DdCBE-DddA.N_ND5.1R_MTS.PD_rep1


title: Amplicon negative control library 2 [CBS1_21]

city: Austin

first: Can

last: Cenik

state: TX

address: Moffett Molecular Biology Building 2.122 2500 Speedway

department: Molecular Biosciences

institute: The University of Texas at Austin

description:
protocol: Total RNA was extracted from HEK293T cells with Qiazol (Qiagen, 79306). 500 ng total RNA was primed with 125 ng random hexamers (Thermo Fisher Scientific, N8080127) and 100 nM final custom oligodT primer (dT30VN) at 65 ˚C for 5 min. Primed RNA was included in a 20 uL RT reaction with 200 U SuperScript III (Thermo Fisher Scientific, 18080051) and incubated at 42˚C for 30 min, 50˚C for 30 min, and 70˚C for 15 min. Reactions were treated with 2.5 U RNaseH (NEB, M0297) at 37 ˚C for 20 min. Each of fifteen CBS tiled amplicons were amplified from 1st strand cDNA with a 25 µL NEB Q5 PCR (NEB, M0491) including 500 nM primers, high GC enhancer, and the following cycling: 98˚C for 30 s; 98 ˚C for 10 s, 62 ˚C for 20 s, 72 ˚C for 30s (18 cycles); final extension at 72 ˚C for 2 min. 22 µL PCR1 product from each tile were pooled and purified by Macherey-Nagel Nucleospin Gel and PCR Cleanup Kit (Takara, 740609) and eluted in 15 µL buffer. 40 ng pooled PCR1 product was input to each of two 50 µL NEB Q5 PCRs with high GC enhancer and NEBNext Multiplex Oligos for Illumina Dual Index Primers Set 1 (NEB, E7600S), with the following cycling conditions: 98˚C for 30 s; 98 ˚C for 10 s, 65 ˚C for 20 s, 72 ˚C for 30 s (4 and 6 cycles); final extension at 72 ˚C for 2 min. Product from the 4 and 6 cycle reactions were pooled and size selected with the Sage Bioscience BluePippin 2% gel (marker M1) with 260-340 bp range. Eluate was concentrated with 1.8x AMPure XP beads (Beckman Coulter, A63881) and eluted in 15 µL water.

satmut_utils parameters: -r CBS_pEZY3.fa -p CBS_pEZY3_primers_coding.bed call -1 CBS1_21_S10_L001_R1_001.fastq.gz -2 CBS1_21_S10_L001_R2_001.fastq.gz --r1_fiveprime_adapters TACACGACGCTCTTCCGATCT,CAAGTTTGTACAAAAAAGTTGGC --r1_threeprime_adapters AGATCGGAAGAGCACACGTCT,CCAACTTTCTTGTACAAAGTGGT --r2_fiveprime_adapters AGACGTGTGCTCTTCCGATCT,ACCACTTTGTACAAGAAAGTTGG --r2_threeprime_adapters AGATCGGAAGAGCGTCGTGTA,GCCAACTTTTTTGTACAAACTTG -g CBS_pEZY3.gff -k CBS_pEZY3.fa -m 1 -s NNK

Illumina MiSeq at Stanford Center for Genomics and Personalized Medicine

data_processing:
Data was analyzed with satmut_utils version 1.0.0. Source code and documentation are available at https://github.com/CenikLab/satmut_utils. A brief description of the satmut_utils data processing steps is outlined below. Analysis parameters are indicated in the 'description: satmut_utils parameters' field.
For RACE-like libraries, 8 bp unique molecular index sequences were extracted with UMItools extract using the following parameters: --extract-method=regex -p (?P<umi_1>[ATCG]{%8}) (?P<discard_1>%AACCGCCAGGAGT){e<=%2}. The regular expression is passed to the satmut_utils call --umi_regex argument.
cutadapt was used for adapter and 3' base quality trimming with the following parameters -q 15 -m 16 -O 8. Adapter sequences differed by sequencing library and are noted in the 'description: satmut_utils parameters' field.
Reads were aligned with bowtie2 in paired-end mode with the following parameters: --maxins=1000 --no-discordant --fr --mp 4 --rdg 6,4 --rfg 6,4
satmut_utils base quality masking: base qualities for read segments originating from synthetic primer sequences were masked by setting them to 0. For amplicon libraries, the 5' and 3' termini of both R1 and R2 may be masked. For RACE-like libraries, only the 3' end of R1 and the 5' end of R2 may be masked.
For RACE-like libraries, reads were grouped with UMItools directional adjacency method and the following parameters: --edit-distance-threshold=1 --paired --ignore-tlen --unpaired-reads=discard --unmapped-reads=discard --multimapping-detection-method=X0 --umi-separator=_. satmut_utils consensus deduplication: group IDs from R1 alignment tags were copied to R2s and paired alignments were sorted by tag. R1s and R2s were consensus deduplicated individually using a majority vote (among duplicates) for the base call at each aligned position. Insertions are not retained in generation of consensus reads. Deletions were retained and deletion gaps longer than 10 bases were converted to the unknown base N.
satmut_utils variant calling: Read pairs are iterated over and pairs were filtered if either read had an edit distance <= 10. Variants were only called if mismatches were found in both mates (concordant base calls) and had base qualities >= 30.
Assembly: Data were aligned to custom reference FASTA files depending on the input source and library preparation chemistry (see description: satmut_utils parameters). Reference files and primer sequences are available at https://github.com/ijhoskins/satmut_utils_supplementary/tree/main/reference_files. Reads may be aligned to the CBS transcript ENST00000398165.7 but some reads may not align as they contain vector sequence.
Supplementary files format and content: Text files containing VCF fields with each INFO tag value in a unique column. See https://github.com/CenikLab/satmut_utils/blob/master/docs/satmut_utils_manual.md for details on each field.
Library strategy: amplicon

growth_protocol: HEK293T LLP iCasp9 Blast cells were obtained from Doug Fowler's lab and cultured in Dulbecco’s Modified Eagle Medium (Thermo Fisher Scientific, 11995065) with 10% fetal bovine serum (Gibco, and 1% penicillin-streptomycin (Gibco, 15140122) at 37°C in 5% CO2 atmosphere. Prior to recombination at passage 16, cells were selected for one week with 2 µg/mL Doxycycline (Sigma-Aldrich, D3072) and 10 µM Blasticidin (Gibco, A1113903) to maintain cells with the integrated landing pad. Cell lines were tested for mycoplasma contamination and were consistently found negative.

extract_protocol:
Negative control libraries were generated by methods detailed in the "description: protocol" field. For samples CBS1_68 through CBS1_82, extraction was as follows. gDNA was extracted from approximately 3-5 million cells with the Cell and Tissue DNA Isolation Kit (Norgen Biotek Corp, 24700), including RNaseA treatment and eluting in 200 µL warm elution buffer. For RNA extraction, 15 million cells were solubilized with 1 mL QIAzol (Qiagen, 79306) and 0.2 mL chloroform in 5PRIME Phase-Lock Gel heavy tubes (QuantaBio, 2302830). RNA was precipitated at -20 ˚C following the addition of 2 µL GlycoBlue (Thermo Fisher, AM9515) and 2.5 volumes of cold absolute ethanol. RNA was washed once with cold 70% ethanol then resuspended in 30 µL water. 10 µg total RNA was treated with DNaseI (NEB, M0303), then re-purified by the RNA Clean and Concentrator kit (Zymo Research, R1015) and eluted in 15 µL water. RNA quality was assessed with the Bioanalyzer Eukaryotic RNA Pico kit (Agilent, 5067-1513).
See library input source- and chemistry-specific preparation details in the "description: protocol" field. For samples CBS1_68 through CBS1_70 and CBS1_79 through CBS1_81, 1st strand cDNA was synthesized as follows: for each of six 20 µL reverse transcription reactions, 2.5 µg DNaseI-treated total RNA was denatured at 65 ˚C for 5 min followed by RT primer annealing at 4 ˚C for 2 min, using 2 pmol RT primer specific for the landing pad (pDEST_HC_Rec_Bxb_v2_R: GGCAGTCTCTCTGATTCCACAGTCAT).  Primed total RNA was included in six 20 µL SuperScript IV cDNA synthesis reactions (Thermo Fisher, 18090010) with SUPERase-In RNase inhibitor (Thermo Fisher, AM2696), and first-strand cDNA was synthesized by incubating at 55 ˚C for 1 h, followed by RT inactivation at 80 ˚C for 10 min. RNA was digested with addition of 5 U RNaseH (NEB, M0297) to the first strand cDNA synthesis reaction and incubation at 37 ˚C for 20 min. One reaction was saved for amplicon library preparation, while the other five were pooled for RACE-like (Anchored Multiplex PCR) library preparation. See library-specific preparation methods in the "description: protocol" field. Unless otherwise noted, NEB Q5 polymerase with high GC enhancer was used in 50 µL PCR reactions with 500 nM primers and the following cycling conditions: initial denaturation at 98 ˚C for 30 s; 3-step cycling with denaturation at 98 ˚C for 10 s, anneal at 65 ˚C for 30 s, extension at 72 ˚C for 1 min; final extension at 72 ˚C for 2 min. Final libraries were quantified using the KAPA Library Quantification Kit for Illumina (Roche, KK4873) according to the manufacturer’s recommendation, using 1:10,000 dilution of libraries and size-correction using an average fragment size of 300 nt. qPCR was performed on the Applied Biosystems ViiA 7 instrument. The final pool was size-selected using Sage Bioscience BluePippin 2% gel (marker V1 and/or V2) to select fragments from 200-400 nt. Following size-selection, the final pool was quantified by both the High Sensitivity DNA Kit (Agilent, 5067-4626), integrating between the 200-600 nt range.

treatment_protocol: See additional details in the "description: protocol" field. For samples CBS1_68 through CBS1_82, treatment was as follows. A CBS variant entry library (Sun and Weile, et al. 2020) was transferred into pDEST_HC_rec_bxb_v2, a vector containing recombination sites for the HEK293T LLP iCasp9 Blast landing pad line, by a Gateway LR II reaction (Thermo Fisher Scientific, 11-791-020) following manufacturer’s recommendations. 1.5 µL of LR reaction was transformed into 25 µL Endura Electrocompetent cells (Lucigen, 60242), plated on Nunc Square Bioassay dishes, scraped in 6 mL LB Miller broth, and 3 mL resuspension was processed with the ZymoPURE II Plasmid Maxiprep kit (Zymo Research, D4203). Library size was estimated at ~540,000 species, or ~30-fold coverage of each possible SNP or MNP variant in the CBS coding sequence. 20 µg of the CBS variant library (in pDEST_HC_Rec_Bxb_V2), along with an equal mass of Bxb1 recombinase (pCAG-NLS-HA-Bxb1) was transfected into three 15 cm dishes of HEK293T LLP iCasp9 Blast cells (passage 18, 65% confluency) using Lipofectamine 3000 (Thermo Fisher Scientific, L3000008), with volumes scaled based on 3.75 µL reagent per 6-well. 24 h later, cells were split 1:2 into 15 cm dishes. 48 h after transfection, 2 µg/mL Doxycycline (Sigma-Aldrich, D3072) and 10 nM AP1903 (MedChemExpress, HY-16046) in DMSO were added for negative selection of non-recombined cells. The next day, dead cells were removed and recombined cells were grown out for an additional two days with addition of fresh media containing Doxycycline and AP1903. Cells were recovered for one day by growth in  media without Doxycycline and AP1903. Transcription was induced with 2 µg/mL Doxycycline for 24 h, cells were stimulated with fresh media for 3 h, and then harvested at 95% confluency.

value: Wild type

tag: molecule

value: Total RNA


title: 293T-DdCBE-Lipo2000-ND5.1-All-PD_rep2

tag: cell type


title: 293T-DdCBE-DddA.N_DddA.C_MTS.PD_rep1

value: treated with TALE-Free_add-MTS


title: Amplicon negative control library 1 [CBS1_10]

description:
protocol: Total RNA was extracted from HEK293T cells with Qiazol (Qiagen, 79306). RNA was DNaseI-treated (Thermo Fisher Scientific, AM2222) and purified with the Direct-zol columns (Zymo Research, R2050). cDNA was synthesized by priming 500 ng total RNA with 100 nM final reverse primer specific to endogenous CBS mRNA (ACGGGAGGGGAAATGATTGCTTCA) at 65 ˚C for 5 min. Primed RNA was included in a 40 uL RT reaction with 200 U SuperScript III (Thermo Fisher Scientific, 18080051) and 20 U SUPERase-In RNase inhibitor (Thermo Fisher, AM2696) and incubated at 55 ˚C for 1 h, then 70 ˚C for 15 min. RNA was degraded by addition of 5 U RNaseH (NEB, M0297) and incubation at 37 ˚C for 20 min. Each of fifteen CBS tiled amplicons were amplified from 50 ng cDNA with a 50 µL Q5 polymerase (NEB, M0491) reaction including 500 nM primers, high GC enhancer, and the following cycling: 98˚C for 30 s; 98 ˚C for 5 s, 62 ˚C for 10 s, 72 ˚C for 20s (18 cycles); final extension at 72 ˚C for 2 min. PCR1 products from each tile were pooled and purified by QIAquick PCR Cleanup Kit (Qiagen, 28104). 20 ng pooled PCR1 product was input to a 25 µL NEB Q5 PCR with high GC enhancer and NEBNext Multiplex Oligos for Illumina Dual Index Primers Set 1 (NEB, E7600S), with the following cycling conditions: 98˚C for 30 s; 98 ˚C for 10 s, 65 ˚C for 20 s, 72 ˚C for 30 s (8 cycles); final extension at 72 ˚C for 2 min. The library was cleaned up with 1.8x AMPure XP beads (Beckman Coulter, A63881).

satmut_utils parameters: -r CBS_pEZY3.fa -p CBS_pEZY3_primers_coding.bed call -1 CBS1-10_S10_L001_R1_001.fastq.gz -2 CBS1-10_S10_L001_R2_001.fastq.gz --r1_fiveprime_adapters TACACGACGCTCTTCCGATCT,CAAGTTTGTACAAAAAAGTTGGC --r1_threeprime_adapters AGATCGGAAGAGCACACGTCT,CCAACTTTCTTGTACAAAGTGGT --r2_fiveprime_adapters AGACGTGTGCTCTTCCGATCT,ACCACTTTGTACAAGAAAGTTGG --r2_threeprime_adapters AGATCGGAAGAGCGTCGTGTA,GCCAACTTTTTTGTACAAACTTG -g CBS_pEZY3.gff -k CBS_pEZY3.fa -m 1 -s NNK

Illumina MiSeq at UT Austin Genome Sequencing and Analysis Facility


title: Lym_58: Xenograft of lymph-node metastasis

city: Chuo-ku

first: Jun

last: Nakayama

state: Tokyo

address: Tsukiji 5-1-1

institute: National Cancer Center

data_processing:
Sequenced reads were checked by FastQC.
Mapping was performed using MiSeq.
The expression matrix was constructed and normlized by Seurat.
Genome_build: GRCh38/hg38

source_name: Xenograft of lymph-node metastasis

growth_protocol: MDA-MB-231 cells were seeded in culture dishes in RPMI1640 medium (gibco)with 10% fetal bovine serum (gibco) with streptomycin and penicillin-G at 37 °C in 5% CO2. Cells were orthotopically transplanted into murine mammary fatpad.

extract_protocol:
Total RNA was extracted  from punched micro-tissue, previously reported (Yoda et al., Sci Rep, 2017).
cDNA libraries for RNA sequencing were

tag: tissue

value: Micro-tissue of Lymph metastasis


title: Lym_57: Xenograft of lymph-node metastasis


title: Lym_59: Xenograft of lymph-node metastasis


title: Lym_55: Xenograft of lymph-node metastasis


title: Lym_61: Xenograft of lymph-node metastasis


title: Lym_54: Xenograft of lymph-node metastasis


title: Lym_56: Xenograft of lymph-node metastasis


title: Lym_50: Xenograft of lymph-node metastasis


title: Lym_53: Xenograft of lymph-node metastasis


title: Lym_70: Xenograft of lymph-node metastasis


title: Lym_69: Xenograft of lymph-node metastasis


title: Lym_43: Xenograft of lymph-node metastasis


title: Lym_42: Xenograft of lymph-node metastasis


title: Lym_52: Xenograft of lymph-node metastasis


title: Lym_60: Xenograft of lymph-node metastasis


title: Lym_49: Xenograft of lymph-node metastasis


title: Lym_48: Xenograft of lymph-node metastasis


title: Lym_71: Xenograft of lymph-node metastasis


title: Lym_45: Xenograft of lymph-node metastasis


title: Lym_27: Xenograft of lymph-node metastasis


title: Lym_19: Xenograft of lymph-node metastasis


title: Lym_44: Xenograft of lymph-node metastasis


title: Lym_17: Xenograft of lymph-node metastasis


title: Lym_20: Xenograft of lymph-node metastasis


title: Lym_16: Xenograft of lymph-node metastasis


title: Lym_41: Xenograft of lymph-node metastasis


title: Lym_15: Xenograft of lymph-node metastasis


title: Lym_11: Xenograft of lymph-node metastasis


title: Lym_18: Xenograft of lymph-node metastasis


title: Lym_12: Xenograft of lymph-node metastasis


title: Lym_47: Xenograft of lymph-node metastasis


title: Lym_03: Xenograft of lymph-node metastasis


title: Lym_13: Xenograft of lymph-node metastasis


title: Lym_08: Xenograft of lymph-node metastasis


title: Lym_07: Xenograft of lymph-node metastasis


title: Lym_14: Xenograft of lymph-node metastasis


title: PT3_95: Xenograft of primary tumor

source_name: Xenograft of primary tumor

value: Micro-tissue of Primary tumor


title: Lym_28: Xenograft of lymph-node metastasis


title: Lym_06: Xenograft of lymph-node metastasis


title: Lym_05: Xenograft of lymph-node metastasis


title: PT3_90: Xenograft of primary tumor


title: Lym_04: Xenograft of lymph-node metastasis


title: Lym_01: Xenograft of lymph-node metastasis


title: PT3_96: Xenograft of primary tumor


title: Lym_09: Xenograft of lymph-node metastasis


title: Lym_10: Xenograft of lymph-node metastasis


title: PT3_91: Xenograft of primary tumor


title: PT3_94: Xenograft of primary tumor


title: PT3_89: Xenograft of primary tumor


title: PT3_80: Xenograft of primary tumor


title: PT3_93: Xenograft of primary tumor


title: Lym_02: Xenograft of lymph-node metastasis


title: PT3_79: Xenograft of primary tumor


title: PT3_88: Xenograft of primary tumor


title: PT3_84: Xenograft of primary tumor


title: PT3_82: Xenograft of primary tumor


title: PT3_85: Xenograft of primary tumor


title: PT3_81: Xenograft of primary tumor


title: PT3_83: Xenograft of primary tumor


title: PT3_78: Xenograft of primary tumor


title: PT3_76: Xenograft of primary tumor


title: PT3_75: Xenograft of primary tumor


title: PT3_71: Xenograft of primary tumor


title: PT3_74: Xenograft of primary tumor


title: PT3_86: Xenograft of primary tumor


title: PT3_73: Xenograft of primary tumor


title: PT3_87: Xenograft of primary tumor


title: PT3_77: Xenograft of primary tumor


title: PT3_92: Xenograft of primary tumor


title: PT3_69: Xenograft of primary tumor


title: PT3_65: Xenograft of primary tumor


title: PT3_64: Xenograft of primary tumor


title: PT3_61: Xenograft of primary tumor


title: PT3_72: Xenograft of primary tumor


title: PT3_62: Xenograft of primary tumor


title: PT3_67: Xenograft of primary tumor


title: PT3_60: Xenograft of primary tumor


title: PT3_68: Xenograft of primary tumor


title: PT3_58: Xenograft of primary tumor


title: PT3_70: Xenograft of primary tumor


title: PT3_66: Xenograft of primary tumor


title: PT3_56: Xenograft of primary tumor


title: sample rep2

city: Hangzhou

first: Yue

last: Li

state: Zhejiang

address: The College of Medicine Keyan C-161, Zijingang Campus, Zhejiang University, Yuhangtang Rd. 866

department: School of medicine

institute: Zhejiang University

data_processing:
low-quality bases and adaptor contamination were trimmed by cutadapt
Filtered reads were mapped to Drosophila genome using BWA mem algorithm
Peaks were called using macs2 callpeak
Genome_build: dm6
Supplementary_files_format_and_content: bigWig files

organism: Drosophila melanogaster

source_name: trachea in white pupae

extract_protocol:
Samples were cross-linked in 1.8% formaldehyde and then were sonicated to produce DNA fragments with an average size of ~500 bp. Sonicated lysates were added to Dynabead protein G coupled with α-Yorkie antibodies and rotated overnight at 4 °C. The chromatin samples were reverse cross-linked at 65 °C for 16 hours.
Immunoprecipitated DNA was prepared for next generation sequencing using the Epicenter Nextera DNA Sample Preparation Kit.

value: trachea

value: white pupae

tag: genotype

value: WT


title: PT3_59: Xenograft of primary tumor


title: PT3_57: Xenograft of primary tumor


title: sample rep3


title: PT3_55: Xenograft of primary tumor


title: sample rep1


title: input


title: PT3_54: Xenograft of primary tumor


title: PT3_63: Xenograft of primary tumor


title: PT3_53: Xenograft of primary tumor


title: RACE-like (Anchored Multiplex PCR) cDNA biological replicate 2 [CBS1_80]

description:
protocol: See growth, treatment, and extract protocol fields. 1st strand cDNA was synthesized according to the Second strand cDNA synthesis was carried out with 1st strand cDNA from each of five reactions converting 2.5 µg total RNA after RNA digestion with RNaseH. 2 pmol landing-pad-specific forward primer (pDEST_HC_Rec_Bxb_v2_F) and 1 µL Q5 polymerase (NEB, M0491) were added to 20 µL of each 1st strand cDNA reaction and incubated as follows: 95 ˚C denaturation for 30 s, followed by three cycles of 1) 55 ˚C anneal for 30 s and, 2) 72 ˚C extension for 3 min, for a total of three linear primer extension cycles. Reactions were pooled and cleaned up with the Nucleospin Gel and PCR Cleanup kit with 1:1 buffer NTI dilution and elution with 15 µL 70 ˚C elution buffer. Double stranded cDNA was fragmented using the Covaris S2 with the following parameters: microTUBE AFA Fiber Snap-Cap, 10% duty cycle, 5 intensity, 200 cycles per burst, 1 min treatment. Sheared ds cDNA inputs were brought up to 50 µL with ultrapure water. Libraries were prepared from 1-1.5 µg sheared ds cDNA with the ArcherDX (now Invitae) High Sensitivity ctDNA Kit according to the manufacturer recommendations (ArcherDX, PRO027.4), using 15 cycles for both PCR1 and PCR2 with custom CBS primers.

satmut_utils parameters: -z -r pDEST_HC_Rec_Bxb_v2_CBS_WT.fa -p pDEST_HC_Rec_Bxb_v2_CBS_WT_primers_coding_GSP2_noendo.bed call -1 CBS1_76_R1.fastq.gz -2 CBS1_76_R2.fastq.gz --r1_fiveprime_adapters AACCGCCAGGAGT --r1_threeprime_adapters AGATCGGAAGAGCACACGTCTGAAC --r2_fiveprime_adapters GTTCAGACGTGTGCTCTTCCGATCT --r2_threeprime_adapters ACTCCTGGCGGTT -g pDEST_HC_Rec_Bxb_v2_CBS_WT.gff -k pDEST_HC_Rec_Bxb_v2_CBS_WT.fa -n 2 -m 1 -s NNK -d

Illumina HiSeq X, NovaSeq 6000 at MedGenome, Inc.

data_processing:
Data was analyzed with satmut_utils version 1.0.0. Source code and documentation are available at https://github.com/CenikLab/satmut_utils. A brief description of the satmut_utils data processing steps is outlined below. Analysis parameters are indicated in the 'description: satmut_utils parameters' field.
For RACE-like libraries, 8 bp unique molecular index sequences were extracted with UMItools extract using the following parameters: --extract-method=regex -p (?P<umi_1>[ATCG]{%8}) (?P<discard_1>%AACCGCCAGGAGT){e<=%2}. The regular expression is passed to the satmut_utils call --umi_regex argument.
cutadapt was used for adapter and 3' base quality trimming with the following parameters -q 15 -m 16 -O 8. Adapter sequences differed by sequencing library and are noted in the 'description: satmut_utils parameters' field.
Reads were aligned with bowtie2 in paired-end mode with the following parameters: --maxins=1000 --no-discordant --fr --mp 4 --rdg 6,4 --rfg 6,4
satmut_utils base quality masking: base qualities for read segments originating from synthetic primer sequences were masked by setting them to 0. For amplicon libraries, the 5' and 3' termini of both R1 and R2 may be masked. For RACE-like libraries, only the 3' end of R1 and the 5' end of R2 may be masked.
For RACE-like libraries, reads were grouped with UMItools directional adjacency method and the following parameters: --edit-distance-threshold=1 --paired --ignore-tlen --unpaired-reads=discard --unmapped-reads=discard --multimapping-detection-method=X0 --umi-separator=_. satmut_utils consensus deduplication: group IDs from R1 alignment tags were copied to R2s and paired alignments were sorted by tag. R1s and R2s were consensus deduplicated individually using a majority vote (among duplicates) for the base call at each aligned position. Insertions are not retained in generation of consensus reads. Deletions were retained and deletion gaps longer than 10 bases were converted to the unknown base N.
satmut_utils variant calling: Read pairs are iterated over and pairs were filtered if either read had an edit distance <= 10. Variants were only called if mismatches were found in both mates (concordant base calls) and had base qualities >= 30.
Assembly: Data were aligned to custom reference FASTA files depending on the input source and library preparation chemistry (see description: satmut_utils parameters). Reference files and primer sequences are available at https://github.com/ijhoskins/satmut_utils_supplementary/tree/main/reference_files. Reads may be aligned to the CBS transcript ENST00000398165.7 but some reads may not align as they contain vector sequence.
Supplementary files format and content: Text files containing VCF fields with each INFO tag value in a unique column. See https://github.com/CenikLab/satmut_utils/blob/master/docs/satmut_utils_manual.md for details on each field.
Library strategy: RACE-like

source_name: HEK293T LLP iCasp9 Blast cells

value: HEK293T LLP iCasp9 Blast

value: Improved landing pad (Matreyek et al., 2020, NAR)


title: PT3_52: Xenograft of primary tumor


title: PT3_50: Xenograft of primary tumor


title: RACE-like (Anchored Multiplex PCR) cDNA biological replicate 3 [CBS1_81]


title: RACE-like (Anchored Multiplex PCR) gDNA biological replicate 1 [CBS1_76]

description:
protocol: See growth and treatment protocol fields. gDNA was fragmented using the Covaris S2 with the following parameters: microTUBE AFA Fiber Snap-Cap, 10% duty cycle, 5 intensity, 200 cycles per burst, 1 min treatment. Sheared DNA inputs were brought up to 50 µL with ultrapure water. Libraries were prepared from 1-1.5 µg sheared gDNA with the ArcherDX (now Invitae) High Sensitivity ctDNA Kit according to the manufacturer recommendations (ArcherDX, PRO027.4), using 15 cycles for both PCR1 and PCR2 with custom CBS primers

satmut_utils parameters: -z -r pDEST_HC_Rec_Bxb_v2_CBS_WT.fa -p pDEST_HC_Rec_Bxb_v2_CBS_WT_primers_coding_GSP2_noendo.bed call -1 CBS1_76_R1.fastq.gz -2 CBS1_76_R2.fastq.gz --r1_fiveprime_adapters AACCGCCAGGAGT --r1_threeprime_adapters AGATCGGAAGAGCACACGTCTGAAC --r2_fiveprime_adapters GTTCAGACGTGTGCTCTTCCGATCT --r2_threeprime_adapters ACTCCTGGCGGTT -g pDEST_HC_Rec_Bxb_v2_CBS_WT.gff -k pDEST_HC_Rec_Bxb_v2_CBS_WT.fa -n 2 -m 1 -s NNK -d

Illumina HiSeq X, NovaSeq 6000 at MedGenome, Inc.

value: Genomic DNA


title: PT3_51: Xenograft of primary tumor


title: RACE-like (Anchored Multiplex PCR) cDNA biological replicate 1 [CBS1_79]


title: RACE-like (Anchored Multiplex PCR) gDNA biological replicate 2 [CBS1_77]


title: Amplicon gDNA biological replicate 3 [CBS1_73]

description:
protocol: See growth, treatment, and extract protocol fields. 2.5 µg of gDNA was amplified with Q5 polymerase (NEB, M0491) for 14 cycles in each of six 50 µL PCR reactions with 500 nM landing-pad-specific primers (CAACTACACGAGCTCACAAGTTTGTACAA, GGCAGTCTCTCTGATTCCACAGTCAT) flanking the entire CBS insert (~1.7 kb), and including the high GC enhancer reagent. The cycling parameters were: initial denaturation at 98 ˚C for 30 s; 3-step cycling with denaturation at 98 ˚C for 10 s, anneal at 65 ˚C for 30 s, extension at 72 ˚C for 1 min; final extension at 72 ˚C for 2 min. Products were pooled, resolved on a 0.8% agarose/TAE gel, visualized with 1x SYBR Gold, and extracted from the gel using the Macherey-Nagel Nucleospin Gel and PCR Cleanup Kit (Takara, 740609) with 25 µL 70 ˚C elution buffer. 500 pg of the PCR1 product was amplified for each of two CBS tiles (2, 4) in a 50 µL NEB Q5 reaction (NEB, M0491) with high GC enhancer for 8 cycles, following the same cycling parameters as for PCR1. CBS tile 2 primers were TACACGACGCTCTTCCGATCTGAAGGGGTCCCCAGAGGATAAG and AGACGTGTGCTCTTCCGATCTTCTGGCAAGATTTTTGGAGATTTTGC. CBS tile 4 primers were TACACGACGCTCTTCCGATCTGAAGTGTGAGCTCTTGGCCAAG  and AGACGTGTGCTCTTCCGATCTCGATCCCGGTGTTCCCGG. Products for tile 2 and 4 amplicons were cleaned up with the Nucleospin Gel and PCR Cleanup Kit (Takara, 740609) and eluted in 30 µL 70 ˚C elution buffer. PCR2 products were mixed together at equal volumes (5 µL each) and input into a final NEB Q5 reaction for 8 cycles with the same conditions using NEBNext Multiplex Oligos for Illumina Dual Index Primers Set 1 (NEB, E7600S) according to manufacturer’s recommendations (65 ˚C annealing). Final library was purified with the Nucleospin Gel and PCR Cleanup kit and eluted in 30 µL 70 ˚C buffer.

satmut_utils parameters: -r pDEST_HC_Rec_Bxb_v2_CBS_WT.fa -p pDEST_HC_Rec_Bxb_v2_CBS_WT_primers_coding_tiles_2_4.bed call -1 CBS1_68_R1.fastq.gz -2 CBS1_68_R2.fastq.gz --r1_fiveprime_adapters TACACGACGCTCTTCCGATCT --r1_threeprime_adapters AGATCGGAAGAGCACACGTCT --r2_fiveprime_adapters AGACGTGTGCTCTTCCGATCT --r2_threeprime_adapters AGATCGGAAGAGCGTCGTGTA -g pDEST_HC_Rec_Bxb_v2_CBS_WT.gff -k pDEST_HC_Rec_Bxb_v2_CBS_WT.fa -n 2 -m 1 -s NNK

Illumina HiSeq X at MedGenome, Inc.


title: Amplicon gDNA biological replicate 2 [CBS1_72]


title: T3

city: Nanning

first: Yinghua

last: Tang

state: Guangxi

address: No. 89-9 Dongge Road

department: Department of Clinical Laboratory

institute: The First Affiliated Hospital of Guangxi University of Chinese Medicine

hyb_protocol: DNA in hybridization solution was denatured at 95℃ for 3 min prior to loading onto a microarray. Arrays were hybridized was preformed in a Agilent Hybridization Oven overnight at a rotation speed of 20 rpm at 42℃ and washed with two consecutive solutions (0.2% SDS, 2× SSC at 42℃ for 5 min, and 0.2× SSC for 5 min at room temperature).

scan_protocol: Scanned on an Agilent G2565CA scanner.

data_processing: Feature Extraction v10.7 (Agilent Technologies, CA) software was used to extract all features of the data obtained from the scanned images and genespring software was used to analyze the raw data, which are normalized by quantile  normalization.

source_name: AMI patient

label_protocol: cDNA labeled with a fluorescent dye (Cy3-dCTP) was produced by Eberwine’s linear RNA amplification method and subsequent enzymatic reaction. This procedure has been previously described, and the procedure has been improved by using CapitalBio cRNA Amplification and Labeling Kit (CapitalBio) for producing higher yields of labeled cDNA.

extract_protocol: RNA was prepared using the TRIzol reagent following the manufacturer's recommendations.  RNA was quantified using a NanoDrop-1000 spectrophotometer.

value: whole blood

tag: Sex

value: female


title: C2

source_name: control

value: male


title: T2


title: Amplicon gDNA biological replicate 1 [CBS1_71]


title: Amplicon cDNA biological replicate 3 [CBS1_70]

description:
protocol: See growth, treatment, and extract protocol fields. 20 µL of 1st strand cDNA reaction was amplified with Q5 polymerase (NEB, M0491) for 14 cycles in a 50 µL PCR reaction with 500 nM landing-pad-specific primers (CAACTACACGAGCTCACAAGTTTGTACAA, GGCAGTCTCTCTGATTCCACAGTCAT) flanking the entire CBS insert (~1.7 kb), and including the high GC enhancer reagent. The cycling parameters were: initial denaturation at 98 ˚C for 30 s; 3-step cycling with denaturation at 98 ˚C for 10 s, anneal at 65 ˚C for 30 s, extension at 72 ˚C for 1 min; final extension at 72 ˚C for 2 min. Products were pooled, resolved on a 0.8% agarose/TAE gel, visualized with 1x SYBR Gold, and extracted from the gel using the Macherey-Nagel Nucleospin Gel and PCR Cleanup Kit (Takara, 740609) with 15 µL 70 ˚C elution buffer. PCR1 was amplified for each of two CBS tiles (2, 4) in a 50 µL NEB Q5 reaction (NEB, M0491) with high GC enhancer for 8 cycles, following the same cycling parameters as for PCR1. CBS tile 2 primers were TACACGACGCTCTTCCGATCTGAAGGGGTCCCCAGAGGATAAG and AGACGTGTGCTCTTCCGATCTTCTGGCAAGATTTTTGGAGATTTTGC. CBS tile 4 primers were TACACGACGCTCTTCCGATCTGAAGTGTGAGCTCTTGGCCAAG  and AGACGTGTGCTCTTCCGATCTCGATCCCGGTGTTCCCGG. Products for tile 2 and 4 amplicons were cleaned up with the Nucleospin Gel and PCR Cleanup Kit (Takara, 740609) and eluted in 30 µL 70 ˚C elution buffer. PCR2 products were mixed together at equal volumes (5 µL each) and input into a final NEB Q5 reaction for 8 cycles with the same conditions using NEBNext Multiplex Oligos for Illumina Dual Index Primers Set 1 (NEB, E7600S) according to manufacturer’s recommendations (65 ˚C annealing). Final library was purified with the Nucleospin Gel and PCR Cleanup kit and eluted in 30 µL 70 ˚C buffer.

satmut_utils parameters: -r pDEST_HC_Rec_Bxb_v2_CBS_WT.fa -p pDEST_HC_Rec_Bxb_v2_CBS_WT_primers_coding_tiles_2_4.bed call -1 CBS1_68_R1.fastq.gz -2 CBS1_68_R2.fastq.gz --r1_fiveprime_adapters TACACGACGCTCTTCCGATCT --r1_threeprime_adapters AGATCGGAAGAGCACACGTCT --r2_fiveprime_adapters AGACGTGTGCTCTTCCGATCT --r2_threeprime_adapters AGATCGGAAGAGCGTCGTGTA -g pDEST_HC_Rec_Bxb_v2_CBS_WT.gff -k pDEST_HC_Rec_Bxb_v2_CBS_WT.fa -n 2 -m 1 -s NNK

Illumina HiSeq X at MedGenome, Inc.


title: C1


title: RACE-like (Anchored Multiplex PCR) gDNA biological replicate 3 [CBS1_78]


title: PT3_48: Xenograft of primary tumor


title: T1


title: Amplicon cDNA biological replicate 2 [CBS1_69]


title: PT3_47: Xenograft of primary tumor


title: 293T-DdCBE-ND5.1-All-PD_rep1

data_processing:
Cut adapter step: The raw FASTQ files are firstly but the sequencing adapters with cutadapt software with the following command: "cutadapt --times 1  -e 0.1  -O 3  --quality-cutoff 25  -m 50 -a  AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC  -A  AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTAGATCTCGGTGGTCGCCGTATCATT -o {read1.out}  -p {read2.out}  {read1.in}  {read2.in}"
Alignment step: After the cut adapter step, all the FASTQ files are mapped to the reference genome, hg38 for HEK293T with bowtie2 aligner. And only record the best hit of alignment for each read.
BAM convert step:The alignment SAM results are converted to sorted BAM files with samtools software
Remove duplication step: The sorted BAM files are removed the PCR amplification bias with the PICARD MarkDuplicates command.
Off-target regions were found using Detect-seq tool(http://detect-seq.com/)
Genome_build: hg38
Supplementary_files_format_and_content: bed file

extract_protocol:
Extracted genomic DNA was fragmented into ~300 bp through Covaris Focused-ultrasonicator™ Instrument (ME220), followed by a purification using DNA Clean & Concentrator-5 (Vistech, DC2005). ~5 ug DNA fragments and 10 pg spike-in model sequences were subjected to end repair with NEBNext End Repair Module (NEB, E6050), and 1 μl E. coli ligase (NEB, M0205) was added to repair nicks in DNA. Then hydroxylamine protection of endogenous 5fC was performed in 100 mM MES buffer (pH 5.0), 10 mM O-ethylhydroxylamine (EtONH2, Aldrich, 274992) at 37 °C for 6 h. The product underwent 5fC protection was diluted 2.5 times and purified with DNA Clean & Concentrator-5. Next, dA-tailing procedure was carried out using NEBNext dA-Tailing Module (NEB, E6053) followed by a DNA purification with 1.8 × AMPure XP beads.  DNA damage that may interfere the subsequent labeling step was repaired in a mixture of 2 μl endonuclease IV (Endo IV, NEB, M0304), 1 μl Bst full-length polymerase (NEB, M0328), 2 μl Taq ligase (NEB, M0208), 1 μl NAD+ (NEB, B9007), 1 μl dNTP (2.5 mM each) in NEBuffer 3 for 1 h at 37 °C and 1 h at 45 °C. DNA was purified and subjected to in vitro BER labeling reaction containing a mixture of 1 μl UDG (NEB, M0280), 1.5 μl Endo IV, 0.8 μl Bst full-length polymerase, 1.7 μl Taq ligase, 1 μl NAD+, 200 nM biotin-dUTP, 800 nM d5fCTP, 200 nM dATP and 200 nM dGTP in NEBuffer 3 for 40 min at 37 °C. The DNA was incubated with 75 mM of malononitrile in 10 mM Tris-HCl (pH 7.0) at 37°C for 20 h in an enclosed thermomixer (Eppendorf, 850 rpm), away from light. The reaction product was then purified with 1.8 × AMPure XP beads.  Labeled fragments were enriched by streptavidin C1 beads (Invitrogen) following the manufacturer's instruction. Y adaptor (see Supplementary Note 1) was ligated (NEBNext Quick Ligation Module, E6056) to double-stranded DNA on streptavidin C1 beads so that free adaptor can be removed by three-times washing of 1×B&W buffer (5 mM Tris-HCl, pH 7.5, 0.5 mM EDTA, 1 M NaCl, 0.05% Tween-20). After washing once with 1× SSC buffer (pH 7.0), beads with biotinylated DNA strands were resuspended in freshly prepared 0.15 M NaOH and incubated at room temperature for 10 min, followed by a sequential washing of 1× SSC buffer (7.0) and 1× TE buffer (pH 7.5) for once. The DNA was eluted from C1 beads using deionized water after heating at 95 °C for 3 min.   Eluted DNA was finally subjected to library amplification with indexed primers (NEB). To obtain sufficient DNA template, 2 rounds of amplification were first performed by using MightyAmp DNA polymerase (Takara), which was proved to read through regions containing biotin-dU and d5fC-malononitrile sites more easily. The PCR product was recovered via DNA Clean & Concentrator-5 and then PCR amplified for ~12 cycles using NEBNext® Q5® Hot Start HiFi PCR Master Mix (NEB, M0543). The constructed libraries were checked using the Agilent 2100 bioanalyzer. Sequencing was performed by Illumina Hiseq X Ten.
Detect-seq

value: HEK293T cells

tag: treatment

value: treated with ND5.1-L1397N


title: Amplicon cDNA biological replicate 1 [CBS1_68]


title: 293T-DdCBE-ND5.1-All-PD_rep2


title: PT3_49: Xenograft of primary tumor


title: PT3_46: Xenograft of primary tumor


title: PT3_42: Xenograft of primary tumor


title: 293T-DdCBE-ND4-All-Input_rep1

description:
WGS
293T-DdCBE-ND4.FinalList.bed.gz

value: treated with ND4-L1397N


title: 293T-DdCBE-ND6-All-PD_rep2

value: treated with ND6-L1397N


title: 293T-DdCBE-GFP-PD_rep1

description: Detect-seq

value: untreated


title: PT3_41: Xenograft of primary tumor


title: 293T-DdCBE-ND6-All-PD_rep1


title: 293T-DdCBE-ND4-All-PD_rep2


title: 293T-DdCBE-ND5.1-All-Input_rep1

description:
WGS
293T-DdCBE-ND5.1.FinalList.bed.gz


title: PT3_44: Xenograft of primary tumor


title: C3


title: 293T-DdCBE-ND4-All-PD_rep1


title: VIR-O pWH1266_1959 2

city: Decatur

first: María

last: Pérez-Varela

state: Georgia

address: 1670 Clairmont Rd

institute: Emory University

data_processing:
Data processing carried out by Genewiz
Assembly: AB5075-UW
Supplementary files format and content: Processed data files contain gene ID, description, log2 expression fold-change and p values.

organism: Acinetobacter baumannii

source_name: VIR-O

growth_protocol: Cultures of Acinetobacter baumannii VIR-O with pWH1266 vector or pWH1266 containing either the ABUW_1959 or ABUW_2818 coding regions driven by the beta-lactamase promoter were grown in 2 mL LB from pure frozen stocks. Grown shaking at 37°C to optical density of 0.7. Cells pelleted and flash-frozen in a dry ice bath and then, stored at -80°C.

extract_protocol:
Pellets were shipped to Genewiz for RNA extraction and rRNA removal.
Library construction using standard Illumina protocols.

tag: phenotype

value: VIR-O

value: 1959 overexpression

value: No treatment

tag: time


title: VIR-O pWH1266_1959 3


title: VIR-O pWH1266_2818 3

value: 2818 overexpression


title: PT3_37: Xenograft of primary tumor

value: Micro-tissue of Primary tumor


title: PT3_40: Xenograft of primary tumor


title: VIR-O pWH1266_2818 1


title: 293T-DdCBE-ND6-All-Input_rep1

description:
WGS
293T-DdCBE-ND6.FinalList.bed.gz


title: WT-stim1

city: New York

first: Laura

last: Campisi

state: NY

address: 1468 Madison Avenue

department: Microbiology

institute: Icahn School Of Medicine at Mount Sinai

description:
CD3/IL2 stimulated; followed by PMA/Ionomycin restimulation CD8 T cells from Mouse 1
A3SS.MATS.JC.txt
A5SS.MATS.JC.txt
MXE.MATS.JC.txt
RI.MATS.JC.txt
SE.MATS.JC.txt

data_processing:
RTA 2.11.3 software was used for base calling. Bcl to Fastq conversion was performed using FASTQ generation pipeline in Illumina Basespace.
For analysis, adaptor removal was performed with cutadapt and base-quality trimming to remove 3′read sequences if more than 20 bases with Q < 20 were present. Paired-end reads were mapped to the mus musculus (mm10) reference genomes with STAR. Alternative splicing analysis was perfomed using rMATS 4.1.081. Downstream splicing predictions was carried out using SPLINTER.
Assembly: mm10
Supplementary files format and content: rMATS output file (Junction Counts only)

source_name: CD8 T Cells

growth_protocol: Splenocytes from 5-6 week old WT animals were dissociated in RPMI 2% FBS, 0.5 mg/ml of Collagenase D and 0.2 mg/ml of DNAse I at 37°C for 30 min with shaking. After red blood cell lysis, CD8 T cell were negatively enriched using a Dynabeads Untouched Mouse CD8 T Cells Kit (Invitrogen), according to the manufacturer’s protocol. After staining, naïve CD62L+ CD44- CD8 T cells were sorted using a BD FACSAria with 5 lasers (BD Bioscience).

extract_protocol:
In vitro activated CD8 T cells were then washed and resuspended in 1 ml TRIzol and isolated following manusfacturer's recomendations. RNA was DNAse-treated using the TURBO DNA-free Kit (Thermofisher scientific). 30ng RNA from the spinal cord samples was used for downstream library preparation.
Libraries were prepared using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB) following manufacturer’s recommendations. Final libraries were quantified and sizing was determined using the High Sensitivity DNA Assay reagents and chip in the Agilent 2100 Bioanalyzer System and the Qubit 1X dsDNA HS Assay Kit respectively.
Individual libraries were then pooled and sequenced using 75bp paired end on the NextSeq 500 using the NextSeq 500/550 High Output Kit.

treatment_protocol: Naïve CD8 T cells (10^5) were resuspended in complete RPMI containing 4 mg/ml of purified anti-mouse CD28 (clone 37.51, Biolegend) and stimulated in 48 well plates coated with 20 mg/ml of purified anti-mouse CD3 (clone 2C11, Biolegend) in an incubator at 37ºC and 5% CO2. Recombinant human IL-2 (10 U/ml, R&D) was added to the culture at the end of day 1. After 3 days of culture, cells were left untreated or washed and additionally restimulated for 1 hour using a Cell Stimulation Cocktail, PMA/Ionomycin (Biolegend).

tag: stimulation

value: 2 rounds of stimulation

tag: paired-samples

value: F


title: WT-stim2

description:
CD3/IL2 stimulated; followed by PMA/Ionomycin restimulation CD8 T cells from Mouse 2
A3SS.MATS.JC.txt
A5SS.MATS.JC.txt
MXE.MATS.JC.txt
RI.MATS.JC.txt
SE.MATS.JC.txt

value: M


title: WT-1

description:
CD3/IL2 stimulated CD8 T cells from Mouse 1
A3SS.MATS.JC.txt
A5SS.MATS.JC.txt
MXE.MATS.JC.txt
RI.MATS.JC.txt
SE.MATS.JC.txt

value: 1 round of stimulation


title: VIR-O pWH1266_1959 1


title: VIR-O pWH1266 vector 3

description:
VIR-O_1266_vs_1959.xlsx
VIR-O_1266_vs_2818.xlsx

value: WT


title: PT3_39: Xenograft of primary tumor


title: VIR-O pWH1266 vector 1


title: PT3_38: Xenograft of primary tumor


title: PT3_32: Xenograft of primary tumor


title: PT3_31: Xenograft of primary tumor


title: WT 2

description:
RNA from bulk spinal cord samples
A3SS.MATS.JC.txt
A5SS.MATS.JC.txt
MXE.MATS.JC.txt
RI.MATS.JC.txt

source_name: Spinal Cord

extract_protocol:
After intracardiac perfusion, spinal cords from 10-12 month old KI and WT mice were dissociated in Hank’s Balanced Salt Solution (HBSS) medium (Gibco), 1 mg/ml of DNAse I and 2 mg/ml of papain (Worhington) at 37°C for 30 min with mild shaking. Tissue suspensions were then filtered through a 70-mm cell strainer. Cell debris were eliminated after resuspension in HBSS 3.5% Bovine Serum Albumin (BSA, Sigma) and centrifuged at 120 g for 10 min. WT1, WT2, WT3, KI1, KI2 and KI3 were isolated as a single batch, while WT4 was isolated on a separate date. Cell pellets were resuspended in HBSS, layered over a 1.06 g/ml solution of Optiprep (Sigma). After centrifugation at 900g for 20 min, cells were collected in the upper layer, washed and resuspended in 1 ml of TRIzol.
RNA from bulk spinal cord samples were isolated using Trizol, following manusfacturer's recomendations. RNA was DNAse-treated using the TURBO DNA-free Kit (Thermofisher scientific). 300ng RNA from the spinal cord samples was used for downstream library preparation.
Libraries were prepared using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB) following manufacturer’s recommendations. Final libraries were quantified and sizing was determined using the High Sensitivity DNA Assay reagents and chip in the Agilent 2100 Bioanalyzer System and the Qubit 1X dsDNA HS Assay Kit respectively. Individual libraries were then pooled and sequenced using 75bp paired end on the NextSeq 500 using the NextSeq 500/550 High Output Kit.

value: bulk spinal cord

tag: isolation day


title: KI 2

value: Setx L389S +/-


title: PT3_33: Xenograft of primary tumor


title: PT3_36: Xenograft of primary tumor


title: WT 1


title: PT3_34: Xenograft of primary tumor


title: WT-3

description:
CD3/IL2 stimulated CD8 T cells from Mouse 3
A3SS.MATS.JC.txt
A5SS.MATS.JC.txt
MXE.MATS.JC.txt
RI.MATS.JC.txt
SE.MATS.JC.txt


title: WT-stim3

description:
CD3/IL2 stimulated; followed by PMA/Ionomycin restimulation CD8 T cells from Mouse 3
A3SS.MATS.JC.txt
A5SS.MATS.JC.txt
MXE.MATS.JC.txt
RI.MATS.JC.txt
SE.MATS.JC.txt


title: WT-2


title: PT3_35: Xenograft of primary tumor


title: PT3_29: Xenograft of primary tumor


title: VIR-O pWH1266 vector 2


title: WT 4


title: VIR-O pWH1266_2818 2


title: KI 1


title: WT 3


title: PT3_27: Xenograft of primary tumor


title: PT3_30: Xenograft of primary tumor


title: RNA-seq_∆WAPL_12h_batch2-rep2

first: Xavier

last: Darzacq

address: 475D Li Ka Shing Center

department: Molecular and Cell Biology

institute: University of California, Berkeley

description: Homozygous cell line with endogenous WAPL loci tagged with an auxin-inducible degron

data_processing: no processed data provided

source_name: Embryonic stem cells

growth_protocol: JM8.N4 mESCs were grown on gelatin-coated plates in knock-out DMEM supplemented with 15% FBS, non-essential amino acids, glutamax, Penicillin-streptomycin, 2-mercapoethanol and LIF. mES cells were fed daily and passaged every 2-3 days by trypsinization.

extract_protocol:
RNAs were extracted using the standard TRIzol RNA extraction protocol and then depleted rRNA with NEBNext rRNA Depletion Kit (NEB, #E6310).
The rRNA-depleted RNAs were then subjected to the RNA-seq library construction using the protocol of NEBNext® Ultra II Directional RNA Library Prep Kit for Illumina® (NEB, #E7765).

treatment_protocol: Degron cell lines were treated for 3 hours with either ethanol only (control, untreated) or with 500 uM, ethanol-dissolved auxin.

value: Embryonic stem cells

value: FLAG-Halo-CTCF / RAD21-SNAPf-V5 / HA-BFP-mAID-WAPL / osTir

value: 500 mM auxin in ethanol, 3 hrs

tag: chip antibody


title: miRNA_AtheroscleroticBrainIschemia#107_rep1

city: Showa-ku, Nagoya

first: Kinya

last: Yokoyama

state: Aichi

address: Tsurumai 65

department: Neurosurgery

institute: Nagoya University Hospital

data_processing:
MiRNA sequencing using 75bp single-read protocol by NextSeq next-generation sequencer (Illumina, USA) was performed at Macrogen Japan Corp. (Tokyo, Japan).
The obtained FASTQ data was uploaded to the geneglobe.qiagen.com website (https://geneglobe.qiagen.com/us/analyze, QIAGEN, USA) for primary data analysis.
The obtained read data was subjected to data normalization and differential expression analysis using the DESeq2 (version 1.28.1) package in the R software (version 4.0.2; http://www.r-project.org/).

source_name: Intracranial cerebrospinal fluid

extract_protocol:
Total RNA extraction from the isolated EVs was performed using miRNeasy Micro Kit (Qiagen, Germany) according to the manufacture's protocol.
One nanogram of miRNA from each sample was used for small RNA library creation using QIAseq miRNA Library Kit (QIAGEN, Germany) according to the manufacturefs protocol.

value: cerebrospinal fluid

tag: gender

value: Female

tag: disease

value: atherosclerotic brain ischemia


title: RNA-seq_∆WAPL_24h_batch2-rep1


title: PT3_26: Xenograft of primary tumor


title: miRNA_AtheroscleroticBrainIschemia#124_rep1

value: Male


title: PT3_25: Xenograft of primary tumor


title: miRNA_AtheroscleroticBrainIschemia#103_rep1


title: scTCR-Seq ALS4 1 to 3 and Controls 1 to 3

description:
TCR seq (scRNA seq)
filtered_contig_annotation_sample_annotations.csv
clonetypes.csv
meta_fastq_submission.csv
meta_features_submission.csv
meta_sample.csv

data_processing:
Illumina RTA3 and RTA2 software were used for base calling scRNA seq and scTCR seq, respectively.
Base call files were demultiplexed using cellranger mkfastq v3.1.0.
scRNA reads were aligned to the GRCh38-3.0.0 genome assembly. scTCR reads were aligned to GRCh38-alts-ensembl-2.0.0.
scRNA gene expression matrices were generated using cellranger count v3.1.0.
V(D)J sequences and annotations were generated using cellranger vdj v3.1.0.
Gene expression matrices were loaded into a single Seurat object using Seurat v4.0.1.
V(D)J sequences and annotations were integrated with the scRNA Seurat object using scRepertoire v1.1.4.
Genome_build: GRCh38-3.0.0 and GRCh38-alts-ensembl-2.0.0
Supplementary_files_format_and_content: For scRNA seq: Cell barcodes, genes, and expression matrices (tar.gz). For scTCR seq: High-level annotations for high-confident cellular contigs (csv) and high-level description of each clonotype (csv).

source_name: PBMC

extract_protocol:
From each patient, blood was drawn into a Cell Preparation Tube with sodium heparin (BD Vacutainer). Peripheral blood mononuclear cells (PBMCs) were isolated by density centrifugation using Ficoll (Ficoll Hystopaque; Sigma) according to the manufacturer’s protocol. PBMCs were aliquoted and frozen at -80°C in FBS (Gibco) containing 10% DMSO (sodium dodecyl sulfate, Sigma). Frozen vials of PBMCs were thawed and washed with culture medium (i.e. RPMI supplemented with 10% FBS, 100 μg/ml penicillin, 100 μg/ml streptomycin, 2 mM L-glutamine, 10 mM HEPES, and 1 nM sodium pyruvate) containing 2 mg/ml of DNAse I (Roche) (45). Cells were resuspended at the density of 2×106/ml in medium containing 0.1 mg/ml of DNAse I and left in culture for 18 hours in an incubator at 37ºC and 5% CO2 before analysis. PBMCs were incubated on ice for 20 min with 100 ul of the hash mixture in PBS 2% Bovine Serum Albumin (BSA, SIGMA) and then washed three time before CD8 T cell negative selection. human CD8 T cells were purified by negative selection  using a Dynabeads Untouched Human CD8 T Cells Kit (Invitrogen), according to the manufacturer’s protocol.Hashed cells were counted on a Nexcelom Autocounter 2000 using AO/PI dye and pooled based on live cell counts. Pooled samples were centrifuged then subsequently resuspended in 100 l of BioLegend’s Total Seq C CITE-Seq antibody panel and incubated on ice for 30 minutes. Antibodies were used at a concentration of 1:100 and diluted in wash buffer (PBS, 0.5% BSA, 1mM EDTA). After 3 washes, cells were resuspended in loading buffer (PBS, 0.5% BSA) and counted. Cells were loaded on one lane of the 10x 5’v1.1 assay with a targeted cell recovery of 20,000 cells.The following antibodies were used for CITE-seq and purchased from Biolegend:  TotalSeq-C0046 anti-human CD8   TotalSeq-C0049 anti-human CD3   TotalSeq-C0063 anti-human CD45RA   TotalSeq-C0148 anti-human CD197 (CCR7)   TotalSeq-C0390 anti-human CD127 (IL-7Rα)   TotalSeq-C0088 anti-human CD279 (PD-1)   TotalSeq-C0154 anti-human CD27   TotalSeq-C0386 anti-human CD28   TotalSeq-C0140 anti-human CD183 (CXCR3)   TotalSeq-C0071 anti-human CD194 (CCR4)   TotalSeq-C0141 anti-human CD195 (CCR5)   TotalSeq-C0143 anti-human CD196 (CCR6)   TotalSeq-C0160 anti-human CD1c   TotalSeq-C0085 anti-human CD25   TotalSeq-C0171 anti-human/mouse/rat CD278 (ICOS)   TotalSeq-C0410 anti-human CD38   TotalSeq-C0087 anti-human CD45RO   TotalSeq-C0168 anti-human CD57   TotalSeq-C0159 anti-human HLA-DR   TotalSeq-C0089 anti-human TIGIT (VSTM3)   TotalSeq-C0250 anti-mouse/human KLRG1 (MAFA
For scRNA seq and scTCR seq, base call files from a NovaSeq6000 Sequencer (Illumina) and a NextSeq550 Sequencer (Illumina), respectively, were generated by the Human Immune Monitoring Center and the Genomic CoRE at the ISMMS.  BCL files were base-called and demultiplexed using cellranger mkfastq v3.1.0. Alignment of gene expression libraries to human reference GRCh38 as well as the counting of unique features of CITESeq libraries were performed using cellranger count v3.1.0.

value: CD8 T cells

tag: library type

value: VDJ TCR seq