title: ATAC-Seq_hTERT-HME1-2

source_name: mammary gland

value: Immortalized with hTERT


title: ATAC-Seq_hTERT-HME1-1

status: Public on Jul 23 2020

type: SRA

city: Stanford

first: Fabian

last: Grubert

address: 300 Pasteur Drive

country: USA

institute: Stanford University

library_source: genomic

data_processing:
we obtained on average 13 ± 7 million paired-end reads
Adaptor sequences were trimmed using cutadapt in paired-end mode (non-default parameters: -q 15 -O 5 -m 30)
Reads were aligned to hg19 using bowtie (version 0.12.8; non-default parameters: -X 2000, -m 1) after which duplicate reads were removed using Picard MarkDuplicates.
Genome_build: hg19
Supplementary_files_format_and_content: bigWig

molecule: genomic DNA

organism: Homo sapiens

growth_protocol: All cell lines were cultured following the manufacturer’s recommendations

extract_protocol: ATAC-seq was carried out as previously described (Buenrostro 2013) and sequenced on an Illumina HiSeq 4000 with 2x100 paired end sequencing.

tag: disease

tag: germ layer

value: Ectoderm


title: 1183T3_Runx1_KO_sg4_rep1

city: Cambridge

first: Lindsay

middle: M

last: LaFave

state: MA

address: 77 Massachusetts Avenue; 76-453

department: Biology

institute: MIT

data_processing:
Base calls were converted to fastq format using bcl2fastq. Raw sequencing reads were trimmed using custom python scripts to remove adapter sequences. The reads were aligned to hg19 or mm10 genome using Bowtie2 (Langmead et  al. 2012) with maximum fragment length set to 2 kb, and all other default settings (bowtie2 -X2000 --rg-id). The data were demultiplexed tolerating one mismatched base within barcodes. Mitochondrial, discordant and low quality reads were removed using SAMtools (Li et al. 2009) (samtools view -b -q 30 -f 0x2). Duplicate sequences were removed using the picard toolkit (http:// broadinstitute.github.io/picard/). Profiles for all cells were first merged into a single alignment (.bam) file and used as input for peak calling with MACS v2.1.2 (MACS2) (Zhang et al., 2008). All default options were used, with the following flags explicitly set: --nomodel, --nolambda, --keep-dup all, --call-summits. This returned a list of single base pair peak summits with associated significance scores (corresponding to log FDR q-value from MACS2). Only peak summits with FDR < 0.01 were retained. Next, a previously described iterative filtering approach was implemented to obtain a list of significant, non-overlapping fixed-width peak windows (Lareau et al., 2019). Briefly, the called peak summits were first padded with 150 base pairs (bp) at either end to generate evenly sized 301 bp window peak regions. Peaks were then sorted in decreasing order of their significance scores. Keeping the most significant peak, overlapping peak windows that had lower significance scores were identified and then removed. This was repeated for the next most significant peak window. Through this iterative process, lower significance overlapping peak regions were filtered out, resulting in 285,956 disjoint 301 bp peak windows.
Genome_build: mm10
Supplementary_files_format_and_content: count files are tab-delimited .txt files of reads in peaks across samples in sparse matrix format. Row names for peaks can be pulled from .bed peak file.
Supplementary_files_format_and_content: bam files for each individual sample are provided following peak calling and data processing
Supplementary_files_format_and_content: .bed peak file, can be used to find peaks for CRISPR data

organism: Mus musculus

source_name: 1183T3; tumor-derived lung cell line

extract_protocol:
Individual tumors were dissected, digested in an enzymatic buffer (1X HBSS, 5mM HEPES, DNaseI, Collagenase IV), and incubated with rotation at 37°C for 30 minutes. The enzymatic buffer was quenched with DMEM and spun at 1000 rpm. Cell pellets were resuspended in DMEM and plated in 6-well plates to allow for attachment. Cell lines were genotyped for Kras, p53, and tomato after 5 passages in culture. The cell lines used in this study were established from mouse LUAD over the course of the study. All lines were grown in DMEM, 10% FBS, and 1% pen-strep. KP cell lines have not been authenticated because the cell lines are not found in established databases. The KP cell lines were tested for mycoplasma and found to be negative. GM12878 cells were grown in DMEM, 10% FBS, and 1% pen-strep and 3T3 cells were grown in RPMI 1640, 10% FBS, and 1% pen-strep. GM12878 cells were authenticated by STR Profiling Service from ATCC.
For CRISPR knockout experiments, guides were cloned into the lentiCRISPR-V2 lentiviral vector (Joung et al., 2017). The lentiCRISPR-V2 vector was digested with Fast Digest EspI and ligated with EspI-compatible annealed oligonucleotides for sgRNAs. KP cell lines were infected with constructs containing guides and selected with puromycin after 48 hours. After puromycin selection, guide performance was tested by western blotting. For CRISPR activation (CRISPRa) experiments, the Lenti-Sam-puro construct was used (developed in the Jacks lab), an adaption of the previously published Lenti-Sam activation construct (Pentinmikko et al., 2019). EspI-compatible cloning was completed and cells were infected with constructs containing guides based on CRISPRa prediction software. Truncated guides of 15 bp were cloned into a lentiviral based expression construct which also encodes for a transcriptional activation complex (MS2-P65-HSF1) and a puromycin selection cassette. Non-metastatic and metastatic cell lines were engineered to express Cas9 following stable selection of a Cas9-Blast construct.

treatment_protocol: Tumor were induced at age 6-10 weeks. Mice were provided Adeno-SPC-Cre virus intratracheally resulting in recombination of Kras G12D, p53, and tdTomato in alveolar type II cells. Cell lines were generated followed by CRISPR perturbation.

tag: strain

value: mixed; C57/BL/6J; 129/SV

tag: tumor stage

value: normal; 25 weeks post-initiation

tag: cell line

tag: treatment


title: ATAC-Seq_HepG2-1

source_name: liver

value: hepatocellular carcinoma

value: Endoderm


title: 1183T3_Runx1_KO_sg5_rep1


title: ATAC-Seq_H1376-1

source_name: urinary bladder

value: carcinoma


title: 1183T3_Runx1_KO_sg3_rep1


title: 1183T3_Runx1_KO_sg5_rep2


title: 1183T3_Runx1_KO_sg2_rep2


title: 1183T3_Runx1_KO_sg2_rep1


title: 1183T3_Runx1_KO_sg3_rep2


title: ATAC-Seq_ARPE-19-2

source_name: retinal pigmented epithelium

value: normal


title: ATAC-Seq_H9-hESC-1

source_name: Embryonic Stem Cell

value: Primitive


title: ATAC-Seq_H1376-2


title: 1183T3_Runx2_KO_sg4_rep3


title: 1183T3_Runx2_KO_sg3_rep2


title: ATAC-Seq_K562-1

source_name: bone marrow

value: chronic myelogenous leukemia

value: Mesoderm


title: ATAC-Seq_H1-hESC-1


title: ATAC-Seq_H9-hESC-2


title: ATAC-Seq_ARPE-19-1


title: 1183T3_Runx3_OE_sg1_rep2


title: ATAC-Seq_H1-hESC-2


title: 1183T3_Runx3_OE_sg1_rep1


title: 1183T3_Runx3_OE_sg2_rep2


title: 1183T3_Runx2_KO_sg3_rep3


title: 1183T3_Runx2_OE_sg2_rep3


title: RNA-Seq_SU-DHL-4-2

data_processing:
FASTQ files were downsampled to 60 million reads
Transcript abundances were  quantified using kallisto (version 0.43.0; non-default parameters: --bias). Transcript sequences (i.e., target sequences) were obtained from Gencode (Release 25; lifted to GRCh37 coordinates).
To visualize RNA-seq data as signal tracks, down-sampled FASTQ files were aligned to the hg19 genome using HiSat2 (version 2.0.5; non-default parameters: -X 1000 --fr --no-mixed --no-discordant)7, after which genome-wide coverage tracks were produced using bedtools (bedtools genomecov -bga -split -ibam). Coverage values were scaled by a constant factor (109/total # of reads) to account for differences in sequencing depth.
Genome_build: hg19

source_name: B lymphocyte; metastatic to peritoneal effusion

extract_protocol:
RNA samples were extracted using the Qiagen All-Prep kit per manufacturer instructions.
Libraries were prepared from total RNA using the TruSeq Stranded Total RNA Library Prep Kit per manufacturer instructions. All libraries were sequenced on the Illumina Hiseq 4000

value: large cell lymphoma


title: RNA-Seq_SU-DHL-2-2

source_name: Lymph node; metastatic to peritoneal effusion

value: large cell lymphoma; histiocytic


title: RNA-Seq_SU-DHL-4-1


title: 1183T3_Runx1_KO_sg4_rep2


title: 1183T3_Runx2_OE_sg2_rep1


title: 1183T3_Cas9_Control_rep5


title: CG14: D4C9-sgFbxw7#12 + Ifng

city: Boston

first: Rizwan

last: Haq

address: 450 Brookline Avenue

department: Medical Oncology

institute: Dana-Farber Cancer Institute

library_source: transcriptomic

data_processing:
Illumina Bcl2fastq v2.18
Raw reads were mapped or aligned to the reference organism using STAR software.
Sequencing reads were aligned to mm9 reference genome using STAR (v2.5)
Raw gene counts were generated from STAR output and normalized RPKM counts were generated using cufflinks v2.2.1
Differential gene expression analysis was performed using both limma and DESeq2.
Genome_build: KEGG analysis was performed using BioJupies software
Supplementary_files_format_and_content: Matrix table with normalized RPKM counts for every gene and every sample

molecule: total RNA

source_name: Ifng (10ng/mL) for 24h

growth_protocol: D4M3A cells were grown in DMEM supplemented with 5%FBS and penicillin–streptomycin

extract_protocol:
Total RNA was extracted using the RNeasy Plus Mini Kit (Qiagen) following the manufacturer’s protocol.
Libraries were prepared using Kapa mRNA Hyper Prep reagent kits from 100ng of purified total RNA according to manufacturer's protocol

treatment_protocol: Cells were treated with Ifng (10ng/mL) or vehicle for 24h

tag: tissue

value: Murine melanoma cells

value: D4M3A-Cas9 melanoma

tag: cell passage


title: RNA-Seq_NP-2

source_name: Neural progenitor


title: RNA-Seq_NP-1


title: 1183T3_Runx3_OE_sg2_rep3


title: 1183T3_Runx2_OE_sg2_rep2


title: 1183T3_Runx3_OE_sg2_rep1


title: ETP_rep1

data_processing:
scATAC-seq paired-end reads were first debarcoded using the bap-barcode utility as part of the bead-based ATAC-seq processing (BAP) pipeline (v0.5.9i; https://github.com/caleblareau/bap), allowing for 1 base mismatch. The resulting sequencing read files were aligned to the mm10 mouse reference genome assembly using BWA v0.7.15, and the corresponding alignment files processed to handle droplet bead multiplet merging using bap, with the following parameter specifications: -r mm10 -bf 500 -bt XB. The same peak set derived using the mouse lung sciATAC-seq data was used to generate a reads in peaks counts matrix for ETP cells.
Genome_build: mm10
Supplementary_files_format_and_content:
count files are three-column tab-delimited .txt files of reads in peaks for single cells in sparse matrix format. First and second columns correspond to the peak and cell barcode ID number, respectively, and the third column having the corresponding count value. Matrix can be read directly using the readMM function as part of the Matrix package in R.
colData file is a tab-delimited (.txt) file containing cell-specific barcodes to demultiplex pooled sequence read files. Columns include CellID (Experiment name and cell barcode tag separated by "_"), BarcodeID (cell barcode tag), and FRIP (indicating the fraction of total uniquely aligned reads that are in peaks)
fragments files are tab-delimited (.tsv) files containing coordinates (chromosome, start, and end) of uniquely aligned fragments, and their associated cell barcode IDs
barcodeTranslate files are tab-delimited (.tsv) files containing the cell barcode IDs and sequences considered by BAP

source_name: scATAC tumor burdened lung

extract_protocol:
Mice were euthanized 8 weeks after tumor initiation in an isoflurane chamber. Lungs were inflated by injecting digestion buffer (Adv DMEM/F12, 5uM HEPES, DNase, 1mg/mL Collagenase, 0.36mM CaCl2) into the trachea. Lungs were dissociated with fine scissors and proteolytic digestion was performed using the lung digestion buffer. Dissociated cells were incubated at 37° for 1 hour with rotation. Cells were washed in 1x PBS and red blood cells were lysed using ACK lysis buffer (Thermo Scientific) for 3 minutes at room temperature. Cells were filtered using a 100um strainer and stained with DAPI (Sigma-Aldrich) and APC-conjugated CD45 (BD, 559864), CD11b (eBioscience 17-0112-82), CD31 (Biolegend, 102510), Ter119 (BD, 557909), and DAPI (Sigma-Aldrich). Fluorescence-activated cell sorting (FACS) of immuno-stained primary cells was performed using a FACSAria sorter (BD) to isolate tdTomato+; DAPI-; APC- tumor cells for sciATAC-seq.
Library was prepped using the SureCell ATAC-Seq Library Prep Kit (10000106678, Bio-Rad). Tagmented cells were loaded onto the ddSEQ Single-Cell Isolator (12004336, Bio-Rad) and libraries were prepared using the SureCell ATAC-Seq Library Prep Kit (17004620, Bio-Rad) and SureCell ATAC-Seq Index Kit (12009360, Bio-Rad). Bead barcoding and indexing were performed utilizing a C1000 Touch thermal cycler with a 96-Deep Well Reaction Module (1851197, Bio-Rad); with a 96-Deep Well Reaction Module (1851197, Bio-Rad). The PCR conditions for indexing were: 37 °C for 30 min, 85 °C for 10 min, 72 °C for 5 min, 98 °C for 30 s, eight cycles of 98 °C for 10 s, 55 °C for 30 s and 72 °C for 60 s, followed by a single 72 °C extension for 5 min. Products were cleaned up using Ampure XP beads (A63880, Beckman Coulter). Further amplication was completed using PCR conditions: 98 °C for 30 s, seven cycles at 98 °C for 10 s, 55 °C for 30 s and 72 °C for 60 s, followed by a single 72 °C extension for 5 min. PCR products were purified using Ampure beads before proceeding to sequencing.

treatment_protocol: Mice with tumors were induced at age 6-10 weeks. Mice were provided Adeno-SPC-Cre virus intratracheally resulting in recombination of Kras G12D, p53, and tdTomato in alveolar type II cells.

tag: strain background

tag: mouse model

value: KPT model

value: normal; 8 weeks post-initiation

value: tumor-burdened whole lung

tag: cell type

value: Sorted tdTom positive tumor cells


title: T-M4

description:
sciATAC tumor-burdened lung
scATACseq_colData_new.txt
scATACseq_mouse_lung_counts.txt
scATACseq_mouse_lung_peaks.bed

data_processing: Supplementary_files_format_and_content: Excel spreadsheet containing cell and sample-specific barcodes to demultiplex pooled sequence read files in .txt format. Columns include cell number, tissue source,lLouvain cluster, index1, and index2.

source_name: Whole Tumor-Burdened Lung

growth_protocol: 3T3 cells were grown in DMEM + 10% FBS + 1% pen/strep; GM12878 cells were grown in RPMI + 10% FBS + 1% pen/strep

extract_protocol:
Lungs were dissociated with fine scissors and then proteolytic digestion was performed using the Lung Dissociation kit (Miltenyi Biotech) following the manufacturer’s instructions. Dissociated cells were then incubated at 37 degrees C for 20 minutes with rotation, then filtered using a 100-μm strainer. Red blood cells were lysed using ACK buffer (Thermo Scientific) and stained with APC-conjugated CD45 (BD, 559864), CD11b (eBioscience 17-0112-82), CD31 (Biolegend, 102510), Ter119 (BD, 557909), and DAPI (Sigma-Aldrich). Fluorescence-activated cell sorting (FACS) of immuno-stained primary cells was performed using a FACSAria sorter (BD) to isolate tdTomato+; DAPI-; APC- tumor cells for sciATAC-seq. Cells were then processed for sciATAC-Seq profiling following 0.1% fixation.
The PCR reaction was carried out at the following conditions: 72 °C for 5 minutes (extension), 98 °C for 5 min, and then thermocycling at 98 °C for 10 s, 70 °C for 30 s and 72 °C for 1 min. After thermocycling for 5 cycles, we took 5 µl sample from a few randomly selected wells and added 10 µl of PCR cocktail with 0.6x SYBRgreen. The 15 µl reactions were amplified to saturation to determine the number of cycles required for the remaining samples on the plate. Libraries were amplified for 13-14 cycles in total. Libraries were pooled and purified using Qiagen MinElute PCR purification column. The libraries were quantified using KAPA library quantification kit. Libraries were sequenced on the Next-seq platform (Illumina) using a 150-cycle kit (Read 1: 47 cycles, Index 1: 36 cycles, Index 2: 36 cycles, Read 2: 47 cycles).
sciATAC-seq

value: mixed; C57/BL/6J; 129/SV

value: late-stage tumor


title: Normal-1, Normal-2, T-M1Pool, T-M1T1, T-M1T2, T-M1T3, T-M1T4, and M-M1N1

description:
sciATAC normal lung, plucked tumor, and metastasis
scATACseq_colData_new.txt
scATACseq_mouse_lung_counts.txt
scATACseq_mouse_lung_peaks.bed

source_name: Whole Lung, Plucked Tumor, Metastasis

value: normal, late-stage tumor, and metastasis


title: 1183T3_Cas9_Control_rep4

value: normal; 25 weeks post-initiation


title: T-M5, M-M5

description:
sciATAC tumor-burdened lung and metastasis
scATACseq_colData_new.txt
scATACseq_mouse_lung_counts.txt
scATACseq_mouse_lung_peaks.bed

source_name: Whole Tumor-Burdened Lung, Metastasis

value: late-stage tumor and metastasis


title: 1183T3_Cas9_Control_rep1


title: 1183T3_Cas9_Control_rep2


title: Normal-CD45neg

description:
sciATAC normal CD45 depleted lung
scATACseq_colData_new.txt
scATACseq_mouse_lung_counts.txt
scATACseq_mouse_lung_peaks.bed

source_name: Whole Lung

value: normal


title: T-M2, T-M2Pool, T-M2T1, T-M2T2, T-M2T3, T-M3, T-M3Pool, T-M3T1, T-M3T2, T-M3T3

description:
sciATAC whole tumor burdened lung and plucked tumor
scATACseq_colData_new.txt
scATACseq_mouse_lung_counts.txt
scATACseq_mouse_lung_peaks.bed

source_name: Whole Tumor-Burdened Lung, Plucked Tumor


title: RNA-Seq_SU-DHL-2-1


title: MCF7_input DNA_ChIPseq

city: Baltimore

first: Tian-Li

last: Wang

state: MD

address: 1551 Jefferson st.

department: Department of Pathology, Oncology, and Gynecology/Obstetrics

institute: Johns Hopkins University

data_processing:
Basecalling was performed by RTA package within the Genome Analyzer Data Collection Software
Fastq files were generated by CASAVA
Overall sequencing quality was checked with the FastQC script.
Reads were aligned to the human genome with BOWTIE2 v2.3.5.1 using default parameter
Peak calling was performed with MACS v 2.2.6 with default parameters
Genome_build: GRCh37 (hg19)
Supplementary_files_format_and_content: .txt files from ChIP-seq experiments contain peak localisation in a MACS output -bed format.

source_name: MCF7 cells, input DNA

growth_protocol: OVCAR3 and MCF7 cells were grown in PRMI 1640 supplemented with 10% fetal bovine serum (FBS)

extract_protocol:
For ChIP-seq DNA extracton, 1.2×107 cells were treated with 5 mM dimethyl 3,3'- dithiobispropionimidate (Thermo Scientific) for 30 min at 4°C and incubated with quenching buffer (100 mM Tris-HCl, 150 mM NaCl) for 5 min. Treated cells were cross-linked with 1% (vol/vol) formaldehyde for 10 min at room temperature, quenched with 125 mM glycine for 5 min, lysed in lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl, pH 8.0), and sonicated by the Bioruptor (Diagenode) to generate DNA fragments ranging from 200 to 600 bp. The sonicated lysates were diluted with ChIP dilution buffer (0.1% TritonX-100, 2 mM EDTA, 20 mM Tris-HCl pH 7.5, 150 mM NaCl, and 1× protease inhibitor), immunoprecipitated with rotation 6-7 hours at 4°C by 3-4 µg of anti-MYC antibodies, pulled down using Protein A/G DYNAL magnetic beads (40 µl of 1:1 mixture), and eluted by the QIAquick PCR Purification Kit (Qiagen).
ChIP-seq library preparation and sequencing were performed by a NextSeq500 platform with single-end reads of 75 bases

value: human cancer cell line

tag: chip antibody

value: none (input control)


title: OVCAR3_input DNA_ChIPseq

source_name: OVCAR3 cells, input DNA


title: ETP_rep2


title: MCF7_Myc_ChIPseq

source_name: MCF7 cells, Myc ChIP

value: anti-c-Myc (Cat No. 1472-1, Epitomics)


title: OVCAR3_Myc_ChIPseq

source_name: OVCAR3 cells, Myc ChIP


title: C8_calb_bg_24hrs_250ng

city: Nijmegen

first: Mariolina

last: Bruno

address: Geert Grooteplein Zuid 10

department: Experimental Internal Medicine

country: Netherlands

institute: Radboudumc

data_processing:
Quality of the acquired sequencing data was controlled using FastQC tool v0.11.5 (Babraham Bioinformatics) and subsequently followed by the removal of adapter sequences and poly(A) tails with Trim Galore! v.0.4.4_dev (Babraham Bioinformatics) and Cutadapt v1.18
On average ~ 6 million reads per individual library were retrieved. Filtered and trimmed reads were mapped to the human reference genome (hg38/GRCh38) using the STAR aligner v2.6.0a
Less than 1% of all reads were comprised of overrepresented sequences and were uniquely mapped with a median of 4 million reads (74.1%). After generating gene level count data using the HTSeq-count tool v0.11.0 an additional filtering step was performed ensuring the exclusion of several non-coding RNAs, i.e. mtRNA, lincRNA, snRNA, tRNA, miscRNA and snoRNA, in our dataset.
Given the absence of sample replicates, PBMC donors were considered biological replicates. Hence, in the differential gene expression analysis using DESeq2 v1.22.0, including logFold Shrinkage and apeglm, the average PBMC donor response to the different stimuli were compared to their control condition, RPMI. Genes with a fold change of >2 and a p-adjusted value <0.01 were considered differentially expressed genes (DEG)
Genome_build: HG38
Supplementary_files_format_and_content: Text files (.counts.txt) containing expression values in counts for each gene. Files are generated by HTSeq-count.

source_name: Peripheral Blood Mononuclear Cells (PBMCs)

extract_protocol:
At 4 and 24 hours cells were lysed with RLT buffer. Prior to subjection to the RNeasy Mini Kit (Qiagen), lysates were homogenized using a 1 mL syringe with a 0.8 x 15 mm needle. RNA was subsequently extracted following manufacturers’ protocol, including an on-column DNAse digestion using the RNAse-Free DNAse set (Qiagen). Quantification and quality assessment of extracted RNA was performed using the Qubit RNA HS assay (Thermo Fisher Scientific) and Agilent 2200 TapeStation (RNA HS Screentape, Agilent), respectively. The majority of samples subjected to quality assessment revealed an RNA integrity number (RINe) of  8.
Libraries were generated from the extracted RNA using the QuantSeq 3’ mRNA-Seq Library Prep Kit-FWD from Lexogen (Lexogen, Vienna, Austria) in accordance to the manufacturers’ protocol. Three separate preparations were performed, split by PBMC donor, in turn limiting the number of samples to 14 to 18 samples per prep. RNA input was normalized to 100 ng for donor A, and to 250 ng for donors B and C. An aliquot (1:10) of double stranded cDNA libraries was used for quantitative PCR, in turn indicating 17 – 18 cycles as optimal for endpoint PCR (17- donor B; 18 - donors A, C). Accurate quantification and assessment of quality of the generated libraries was performed using Qubit dsDNA HS assay (Thermo Fisher Scientific, Waltham, USA) and Agilent 2200 TapeStation (HS-D1000 ScreenTape, Agilent, Santa Clara, USA). The cDNA concentration and average fragment size were used to determine the molar concentration of the individual libraries. Consequently, libraries were pooled equimolar to 100 fmol. After a final dilution of the pool to a concentration of 4 nM, the libraries were sequenced on a NextSeq 500 instrument (Illumina, San Diego, USA), with 75 cycle (i.e. 75bp single-end sequence reads), high output kit with a 1.1 pM final loading concentration.

tag: time

tag: stimulus

tag: donor

value: Donor C


title: C12_caur_KTClive_24hrs_repl_250ng

value: Peripheral Blood Mononuclear Cells (PBMCs)


title: C14_caur_KTCbg_24hrs_250ng


title: C13_caur_KTCman_24hrs_250ng


title: C10_caur_KTCman_4hrs_250ng


title: C7_calb_man_24hrs_250ng


title: C9_caur_KTClive_4hrs_repl_250ng


title: C6_calb_live_24hrs_250ng


title: C3_calb_man_4hrs_250ng


title: C5_RPMI_24hrs_250ng

value: RPMI


title: C2_calb_live_4hrs_250ng


title: 1183T3_Cas9_Control_rep3

source_name: 1183T3; tumor-derived lung cell line


title: B15_caur_KTCbg_24hrs_250ng

value: Donor B


title: B14_caur_KTCman_24hrs_250ng


title: Mouse-Cortex_HyPBase_scCC

city: St. Louis

first: Robi

middle: D

last: Mitra

state: Missouri

address: 4515 McKinley Ave

department: Genetics

institute: Washington University in St. Louis

description:
Single cell calling cards library
SRRs represent biological replicates.

data_processing:
Read 1 sequenced the cell barcode and unique molecular index of each self-reporting transcript. Read 2 began with GGTTAA (end of the piggyBac terminal repeat and insertion site motif) before continuing into the genome. Reads containing this exact hexamer were trimmed using cutadapt with the following settings: -g "^GGTTAA" --minimum-length 1 --discard-untrimmed -e 0 --no-indels. Reads passing this filter were then trimmed of any trailing P7 adapter sequence, again using cutadapt and with the following settings: -a "AGAGACTGGCAAGTACACGTCGCACTCACCATGANNNNNNNNNATCTCGTATGCCGTCTTCTGCTTG" --minimum-length 1. Reads passing these filters were aligned using 10x Genomics’ cellranger with the following settings: --expect-cells=6000 --nosecondary --chemistry=SC3Pv2 --localcores=16 --localmem=30. This workflow also managed barcode validation and collapsing of UMIs. Aligned reads were validated by verifying that they mapped adjacent to TTAA tetramers. Reads were then converted to calling card format (.ccf.txt). Finally, to minimize the presence of intermolecular artifacts, we required that each insertion must have been tagged by at least two different UMIs. We used the set of validated cell barcodes from each scRNA-seq library to demultiplex library-specific barcoded insertions from the scCC data. This approach requires no shared cell barcodes between individual scCC (and scRNA-seq) libraries. As a result, we excluded insertions from non-unique cell barcodes, which represented a very small number of total cells lost (< 1% per multiplexed library).
Genome_build: mm10
Supplementary_files_format_and_content: BAM files contain aligned and annotated calling card reads. Barcode files contain validated single cell barcodes. ccf.txt files contain processed calling card data. Peaks file provides details on regions significantly enriched for calling card insertions.  describes calling card-specific BAM tags.

molecule: polyA RNA

source_name: Mouse cortex

growth_protocol: All mouse experiments were done following procedures described in (Cammack et al., 2019). In brief, we cloned the PB-SRT-tdTomato and HyPBase constructs into AAV vectors. The Hope Center Viral Vectors Core at Washington University in St. Louis packaged each construct in AAV9 capsids. Titers for each virus ranged between 1.1e13 and 2.2e13 viral genomes/ml. All animal practices and procedures were approved by the Washington University in St. Louis Institutional Animal Care and Use Committee (IACUC) in accordance with National Institutes of Health (NIH) guidelines.

extract_protocol:
Mouse cortical tissues were dissociated to single suspensions following a modification of previously published methods (Avey et al., 2018; Saxena et al., 2012). We incubated samples in a papain solution containing Hibernate-A with 5% v/v trehalose, 1x B-27 Supplement, 0.7 mM EDTA, 70 µM 2-mercaptoethanol, and 2.8 mg/ml papain. After incubation at 37ºC, cells were treated with DNaseI, triturated through increasingly narrow fire-polished pipettes, and passed through a 40-micron filter prewetted with resuspension solution: Hibernate-A containing 5% v/v trehalose, 0.5% Ovomucoid Trypsin Inhibitor, 0.5% Bovine Serum Albumin (BSA), 33 µg/ml DNaseI (Worthington), and 1x B-27 Supplement. The filter was washed with 6 ml of resuspension solution. The resulting suspension was centrifuged for 4 minutes at 250 g. The supernatant was discarded. The pellet was then resuspended in 2 ml of resuspension solution and resuspended by gentle pipetting. We eliminated subcellular debris using gradient centrifugation. We first prepared a working solution of 30% w/v OptiPrep Density Gradient Medium mixed with an equal volume of 1x Hank’s Balanced Salt Solution (HBSS) with 0.5% BSA. We then prepared solutions of densities 1.057, 1.043, 1.036, and 1.029 g/ml using by combining the working solution with resuspension solution at ratios of 0.33:0.67, 0.23:0.77, 0.18:0.82, and 0.13:0.87, respectively. We layered 1 ml aliquots of each solution in a 15 ml conical tube beginning with the densest solution on the bottom. The cell suspension was added last to the tube and centrifuged for 20 minutes at 800g at 12ºC. The top layer was then aspirated and purified cells were isolated from the remaining layers. These cells were then resuspended in FACS buffer: 1x HBSS, 2 mM MgCl2, 2 mM MgSO4, 1.25 mM CaCl2, 1 mM D-glucose, 0.02% BSA, and 5% v/v trehalose. Cells were centrifuged for 4 minutes at 250 g, the supernatant was discarded, and the pellet was resuspended in FACS buffer by gentle pipetting. Cells were then sorted based on fluorescence activity. As a gating control, we analyzed cells from cortices injected with AAV9-PB-SRT-tdTomato only. We then collected cells from brains transfected with AAV9-PB-SRT-tdTomato and AAV9-HyPBase whose fluorescence values exceeded the gate. After sorting, cells were centrifuged for 3 minutes at 250 g. The supernatant was discarded and cells were resuspended in FACS buffer at a concentration appropriate for 10x Chromium 3’ scRNA-seq library preparation. Single cell RNA-seq libraries were prepared using 10x Genomics’ Chromium Single Cell 3’ Library and Gel Bead Kit. Each replicate was targeted for recovery of 6,000 cells. Library preparation followed a modified version of the manufacturer’s protocol. We prepared the Single Cell Master Mix without RT Primer, replacing it with an equivalent volume of Low TE Buffer. Gel-in-emulsion (GEM) generation and GEM-RT incubation proceeded as instructed. At the end of Post GEM-RT cleanup, we added 36.5 µl Elution Solution I and transferred 36 µl of the eluted sample to a new tube (instead of 35.5 µl and 35 µl, respectively). The eluate was split into two 18 µl aliquots and kept at –20ºC until ready for further processing. One fraction was kept for single cell calling cards library preparation, while the other half was further processed into a single cell RNA-seq library.
To amplify self-reporting transcripts from single cell RNA-seq libraries, we took 9 µl of RT product (the other half was kept in reserve) and added it to 25 µl Kapa HiFi HotStart ReadyMix and 15 µl ddH2O. We then prepared a PCR primer cocktail comprising 5 µl of 100 µM Bio_Illumina_Seq1_scCC_10X_3xPT primer, 5 µl of 100 µM Bio_Long_PB_LTR_3xPT, and 10 µl of 10 mM Tris-HCl, 0.1 mM EDTA buffer. One µl of this cocktail was added to the PCR mixture and placed in a thermocycler. Thermocycling settings were as follows: 98ºC for 3 minutes; 20-22 cycles of 98ºC for 20 seconds–67ºC for 30 seconds–72ºC for 5 minutes; 72ºC for 10 minutes; 4ºC forever. PCR purification was performed with 30 µl AMPure XP beads (0.6x ratio) as described previously. The resulting library was quantitated on an Agilent TapeStation 4200 System using the High Sensitivity D5000 ScreenTape. Single cell calling card library preparation was performed using the Nextera Mate Pair Sample Prep Kit with modifications to the manufacturer’s protocol. The library was circularized by bringing 300 fmol (approximately 200 ng) of DNA up to a final volume of 268 µl with ddH2O, then adding 30 µl Circularization Buffer 10x and 2 µl Circularization Ligase (final concentration: 1 nM). This reaction was incubated overnight (12-16 hours) at 30ºC. After removal of linear DNA (following manufacturer’s instructions), we sheared the library on a Covaris E220 Focused-ultrasonicator with the following settings–peak power intensity: 200; duty factor: 20%; cycles per burst: 200; time: 40 seconds; temperature: 6ºC. The library preparation was performed per manufacturer’s instructions until adapter ligation. We designed custom adapters (Table S5) so that the standard Illumina sequencing primers would not interfere with our library. Adapters were prepared by combining 4.5 µl of 100 µM scCC_P5_adapter, 4.5 µl of 100 µM scCC_P7_adapter, and 1 µl of NEBuffer 2, then heating in a thermocycler at 95ºC for 5 minutes, then holding at 70ºC for 15 minutes, then ramping down at 1% until it reached 25ºC, holding at that temperature for 5 minutes, before keeping at 4ºC forever. One microliter of this custom adapter mix was used in place of the manufacturer’s recommended DNA Adapter Index. The ligation product was cleaned per manufacturer’s instructions. For the final PCR, the master mix was created by combining 20 µl Enhanced PCR Mix with 28 µl of ddH2O and 1 µl each of 25 µM scCC_P5_primer and 25 µM scCC_P7_primer. This was then added to the streptavidin bead-bound DNA and amplified under the following conditions: 98ºC for 30 seconds; 15 cycles of: 98ºC for 10 seconds–60ºC for 30 seconds–72ºC for 2 minutes; 72ºC for 5 minutes; 4ºC forever. All of the PCR supernatant was transferred to a new tube and purified with 35 µl (0.7x) AMPure XP beads following manufacturer’s instructions. The final library was eluted in 25 µl Elution Buffer and quantitated on an Agilent TapeStation 4200 System using the High Sensitivity D1000 ScreenTape.

treatment_protocol: We mixed equal volumes of each virus and performed intracranial cortical injections of the mixture into newborn wild-type C57BL/6J pups (P0-2). As a gating control, we injected one litter-matched animal with AAV9-PB-SRT-tdTomato only. After 2 to 4 weeks, we sacrificed mice and dissected the cortex (8 libraries) or hippocampus (1 library).

value: cerebral cortex


title: C1_RPMI_4hrs_250ng


title: RNA-Seq_GM12878-1

source_name: B cell; lymphoblastoid

value: Immortalized with EBV

value: Mesoderm


title: B11_caur_KTCbg_4hrs_250ng


title: B9_caur_KTClive_4hrs_250ng


title: B8_calb_bg_24hrs_250ng


title: C11_caur_KTCbg_4hrs_250ng


title: RNA-Seq_DU145-2

source_name: prostate; metastatic to brain

value: carcinoma

value: Endoderm


title: RNA-Seq_ARPE-19-2

source_name: retinal pigmented epithelium

value: Ectoderm


title: B7_calb_man_24hrs_250ng


title: B10_caur_KTCman_4hrs_250ng


title: RNA-Seq_GM12878-2


title: RNA-Seq_DU145-1


title: RNA-Seq_ARPE-19-1


title: B6_calb_live_24hrs_250ng


title: B5_RPMI_24hrs_250ng


title: B4_calb_bg_4hrs_250ng


title: Scer_WT_mock_rep2

city: Tel Aviv

first: Aldema

last: Sas-Chen

state: Israel

address: Tel Aviv University

department: Shmunis School of Biomedicine and Cancer Research

country: Israel

institute: Tel Aviv University

data_processing:
Library strategy: ac4C-seq
alignment using STAR (V. 2.5.3a)
base-calling using JACUSA v2.0.0 under default parameters
calculation of misincorporation rates per base
Genome_build: ASM996v1 for Thermococcus kodakarensis, ASM27560v1 for Pyrococcus furiosus, ASM15120v2 was used for Thermococcus sp. AM4, ASM700v1 for Sulfolobus solfataricus and ASM9166v1 for Methanocaldococcus jannaschii. For human GRCh37/hg19 with UCSC Genes annotations. For Saccharomyces cerevisiae samples the sacCer3 assembly.
Supplementary_files_format_and_content: SasChen_TableS2.xlsx an excel file detailing ac4C sites identified in each organism including the level of modification of each site.

organism: Saccharomyces cerevisiae

source_name: yeast strain

growth_protocol:
T. kodakarensis and T. sp AM4 strains – TS559 can derivatives thereof – were grown as previously described (Santangelo et al. 2007; Hileman and Santangelo 2012; Gehring et al. 2017) in artificial sea water (ASW) medium supplemented with vitamins and trace minerals. Pyrococcus furiosus strain COM1 was cultured at 75-95˚C in an artificial sea water based medium supplemented with cellobiose, maltose, yeast extract, S˚, trace minerals, cysteine and sodium tungstate as previously described (Lipscomb et al, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Apr. 2011, p. 2232–2238).
HEK-293T and HeLa (WT and Nat10 depleted) cells were grown in DMEM media.
Saccharomyces cerevisiae strains were grown at 30°C in standard YEP medium (1% yeast extract, 2% Bacto Peptone) supplemented with 2% galactose (YPG), or 2% dextrose (YPD).

extract_protocol:
Total RNA was isolated from T. kodakarensis using TRIZOL according to manufacturer’s protocol. For the indicated samples of T. kodakarensis ,rRNAs were depleted according to  Morlan et al.(Morlan et al. 2012) using reagents provided in the NEBNext® rRNA Depletion Kit (NEB #E6310). For indicated samples purifications of T. kodakarensis ribosomes of the wild-type and TkNat10 knockout were conducted similar to previously documented procedures (Matzov et al. 2017).
Total RNA from Human Cells was extracted using TRIZOL according to manufacturer's protocol. For the indicated samples Poly(A) RNA from yeast and human total RNA was isolated by two rounds of purification using the  GenElute mRNA miniprep kit (Sigma) according to manufacturer's protocol.  500ug total RNA was used per purification column.  Typical yield after two rounds of isolation was 1.2%.
Total RNA was isolated from Yeast using hot acidic phenol.  Briefly, frozen yeast (S. cerevisiae) pellet suspended in 1.0 mL AES buffer (50mM NaOAc, 10mM EDTA ph 8.0, 1% SDS) per 0.5mL pellet volume.  To suspended pellet, 1.0 mL Acid buffered Phenol per mL of AES buffer used was added. Sample was mixed by vortexing and incubated in a 65˚C water bath for 30 min, vortexing every 2 minutes to mix.  Samples were put on ice for 10 minutes and 1.0 mL Chloroform:isoamyl alcohol (24:1) was added for each 1.0 mL phenol used. Sample was vortexed to mix and centrifuged 5000 rcf for 15 minutes. Aqueous layer (top) was transferred to a clean tube and extracted 3X with an equal volume of acid buffered phenol:chlorofom:isoamyl alcohol (24:23:1).  After each extraction sample was centrifuged at 5000 rcf for 10 minutes and aqueous layer transferred to a new tube. A final extraction with chloroform:isoamyl alcohol was carried out to remove residual phenol. Aqueous layer was transferred to a clean tube and RNA was precipitated by the addition of an equal volume of 100% isopropanol and 1/9th volume of 3M Sodium Acetate.  Samples were incubated -20˚C for 30 minutes and centrifuged 12000 rcf at 4˚C for 15 minutes. Supernatant was decanted and the pellet washed with 4 mL ice fold 70% ethanol. RNA pellet was resuspended by briefly heating at 50 ˚C in 1.0 mL 1X TE buffer pH 8.0. Samples were quantified by UV absorbance and stored at -80˚C. Typical extractions were carried out with 1.0 mL volume cell pellets and yielded 20mg of total RNA
Fragmentation, 3' adapter ligation, cDNA synthesis, second adapter ligation and enrichment

treatment_protocol: HEK-293T cells transfected with 3xFLAG-tagged NAT10 and myc-tagged THUMPD1 using FuGENE® 6 transfection reagent (Promega, #E2691) 24 hours prior to harvesting.

tag: genetic background

value: WT

value: mock

tag: growth condition

value: 30 degrees


title: B13_caur_KTClive_24hrs_250ng


title: Scer_WT_mock_rep1


title: Scer_Kre33mut_NaCNBH3_rep1

value: Kre33 catalytic mutant


title: Scer_WT_NaCNBH3_rep1


title: Scer_WT_NaCNBH3_rep2


title: Scer_Kre33mut_NaCNBH3_rep2


title: C4_calb_bg_4hrs_repl_250ng


title: B2_calb_live_4hrs_250ng


title: B1_RPMI_4hrs_250ng


title: Scer_OEkre33tan1_YPG_mock

value: Kre33/Tan1 overexpression


title: Scer_OEkre33tan1_YPG_NaCNBH3


title: Scer_OEkre33tan1_YPD_mock


title: Scer_OEkre33tan1_YPD_NaCNBH3


title: A15_caur_KTCbg_24hrs_100ng

value: Donor A


title: A14_caur_KTCman_24hrs_100ng


title: A13_caur_KTClive_24hrs_100ng


title: Scer_WT_YPG_mock


title: Scer_WT_YPD_NaCNBH3


title: A9_caur_KTClive_4hrs_100ng


title: A10_caur_KTCman_4hrs_100ng


title: Scer_WT_YPG_NaCNBH3


title: A11_caur_KTCbg_4hrs_100ng


title: Lib_hP_casp8_R3

city: San Francisco

first: Jim

last: Wells

state: CA

department: Pharmaceutical Chemistry

institute: ucsf

description: Nextseq

data_processing:
Library strategy: SPD-NGS
Raw NGS data from Lib 10AA, “*.fastq.gz” sequencing files were converted into a table with DNA sequences, amino acid sequences, counts/frequency, RPM as four columns, which were saved as .csv files for further analysis.
For Lib hP, reads were aligned to a reference dataset using bowtie2 to generate Sam files. Sam files were then converted into Bam files using Samtools, which were then parsed using a suite of in-house analysis tools (R) to make a table with gene, ref.name, individual phage counts/frequency, peptide sequence, etc. as columns. These tables are saved as .csv files for further analysis.
Supplementary_files_format_and_content: Lib10AA data was processed directly by R script. Lib hP data was first aligned to reference dataset using bowtie2 to generate sam file. The sam files were then converted to bam files using Samtools. Bam files  can be processed using R script.

organism: synthetic construct

source_name: phage

growth_protocol: phage was amplified using XL1-Blue culture

extract_protocol:
We used phage amplification overnight cultures for NGS sample preparation after boiling the phage to extract ssDNA as templates for PCR reactions.
For all samples from both Lib 10AA and Lib hP, a barcoding PCR process was performed using primers listed in Supplemental Figure 21.The thermal PCR profile for Lib 10AA was: 98 °C (20 s), 63 °C (15 s), 72 °C (30 s), 15 cycle. The thermal profile for Lib hP was: 98 °C (30 s), 72 °C (30 s), 15 cycle to prevent heterodimerization due to the 24-residue overlap. The number of cycles was determined empirically to prevent product laddering, assessed by agarose gel electrophoresis. PCR products were gel purified on 1.2 % agarose. Libraries were sequenced on a NextSeq or Highseq4000(Illumina) using single-read 50 or 150 base pair reads. A custom sequencing primer was used (order as shown): ATCGGATCCGCCGCTCTGAAAGTACAGATTCTC (reverse, Tm =67 ◦C, GC% = 52) or CTGCAGTATGTGGCATGAATCTGGTGGTGGT (reverse, Tm =67 ◦C, GC% = 52) for Lib 10AA and GTATGACGTCGCCATCCGCAGTTCGAGAAA (forward, Tm =67 ◦C, GC% = 52) for Lib hp.

treatment_protocol: To perform a screening, 1 mL of biotinylated substrate phage library was incubated with 100 µL magnetic StreptAvidin (SA) beads for 30 min and after that, the SA beads were then stringently washed with PBS buffer supplemented with 0.05% Tween20 and 0.2% BSA to get rid of any free phage. After the SA beads were treated with protease of interest, released phage in supernatant were collected and propagated as protease-sensitive pool, and those bound on the beads can also be propagated as protease-resistant pool after released by non-specific proteases like trypsin. After three rounds of selection, phage polymerase chain reaction (PCR) was conducted, followed by NGS to identify sensitive and resistant substrate sequences in each pool.

tag: fragment size

tag: sample prep

value: phage_PCR and gel extraction


title: A6_calb_live_24hrs_100ng


title: Lib_hP_casp8_R2

description: Nextseq


title: Lib_hP_casp8_R1

description: Nextseq


title: Scer_WT_YPD_mock


title: A3_calb_man_4hrs_100ng


title: Lib_hP_casp7_R3

description: Nextseq


title: A4_calb_bg_4hrs_100ng


title: A5_RPMI_24hrs_100ng


title: Lib_hP_casp7_R1

description: Nextseq


title: A8_calb_bg_24hrs_100ng


title: Lib_hP_casp7_R2

description: Nextseq


title: A1_RPMI_4hrs_100ng


title: Lib_hP_casp6_R3

description: Nextseq


title: A7_calb_man_24hrs_100ng


title: Lib_hP_casp6_R2

description: Nextseq


title: Lib_hP_casp6_R1

description: Nextseq


title: Lib_hP_casp3_R1

description: Nextseq


title: Lib_hP_casp3_R3

description: Nextseq


title: Lib_hP_casp2_R1

description: Nextseq


title: HEK_OEnat10thumpd1_mock_rep2_run1

source_name: human cell line

value: Nat10/Thumpd1 overexpression

value: 37 degrees


title: A2_calb_live_4hrs_100ng

value: Peripheral Blood Mononuclear Cells (PBMCs)


title: Lib10AA_casp8_R2

description: Nextseq


title: HEK_OEnat10thumpd1_NaCNBH3_rep2_run1


title: HCT-116_SP1-HyPBase_scCC

data_processing:
Read 1 sequenced the cell barcode and unique molecular index of each self-reporting transcript. Read 2 began with GGTTAA (end of the piggyBac terminal repeat and insertion site motif) before continuing into the genome. Reads containing this exact hexamer were trimmed using cutadapt with the following settings: -g "^GGTTAA" --minimum-length 1 --discard-untrimmed -e 0 --no-indels. Reads passing this filter were then trimmed of any trailing P7 adapter sequence, again using cutadapt and with the following settings: -a "AGAGACTGGCAAGTACACGTCGCACTCACCATGANNNNNNNNNATCTCGTATGCCGTCTTCTGCTTG" --minimum-length 1. Reads passing these filters were aligned using 10x Genomics’ cellranger with the following settings: --expect-cells=6000 --nosecondary --chemistry=SC3Pv2 --localcores=16 --localmem=30. This workflow also managed barcode validation and collapsing of UMIs. Aligned reads were validated by verifying that they mapped adjacent to TTAA tetramers. Reads were then converted to calling card format (.ccf.txt). Finally, to minimize the presence of intermolecular artifacts, we required that each insertion must have been tagged by at least two different UMIs. We used the set of validated cell barcodes from each scRNA-seq library to demultiplex library-specific barcoded insertions from the scCC data. This approach requires no shared cell barcodes between individual scCC (and scRNA-seq) libraries. As a result, we excluded insertions from non-unique cell barcodes, which represented a very small number of total cells lost (< 1% per multiplexed library).
Genome_build: hg38
Supplementary_files_format_and_content: BAM files contain aligned and annotated calling card reads. Barcode files contain validated single cell barcodes. ccf.txt files contain processed calling card data. Peaks file provides details on regions significantly enriched for calling card insertions.  describes calling card-specific BAM tags.

growth_protocol: Cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% of either penicillin-streptomycin or antibiotic-antimycotic. Cells were grown at 37ºC with 5% carbon dioxide (CO2). Media was replenished every 2 days.

extract_protocol:
Single cell RNA-seq libraries were prepared using 10x Genomics’ Chromium Single Cell 3’ Library and Gel Bead Kit. Each replicate was targeted for recovery of 6,000 cells. Library preparation followed a modified version of the manufacturer’s protocol. We prepared the Single Cell Master Mix without RT Primer, replacing it with an equivalent volume of Low TE Buffer. Gel-in-emulsion (GEM) generation and GEM-RT incubation proceeded as instructed. At the end of Post GEM-RT cleanup, we added 36.5 µl Elution Solution I and transferred 36 µl of the eluted sample to a new tube (instead of 35.5 µl and 35 µl, respectively). The eluate was split into two 18 µl aliquots and kept at –20ºC until ready for further processing. One fraction was kept for single cell calling cards library preparation, while the other half was further processed into a single cell RNA-seq library.
To amplify self-reporting transcripts from single cell RNA-seq libraries, we took 9 µl of RT product (the other half was kept in reserve) and added it to 25 µl Kapa HiFi HotStart ReadyMix and 15 µl ddH2O. We then prepared a PCR primer cocktail comprising 5 µl of 100 µM Bio_Illumina_Seq1_scCC_10X_3xPT primer, 5 µl of 100 µM Bio_Long_PB_LTR_3xPT, and 10 µl of 10 mM Tris-HCl, 0.1 mM EDTA buffer. One µl of this cocktail was added to the PCR mixture and placed in a thermocycler. Thermocycling settings were as follows: 98ºC for 3 minutes; 20-22 cycles of 98ºC for 20 seconds–67ºC for 30 seconds–72ºC for 5 minutes; 72ºC for 10 minutes; 4ºC forever. PCR purification was performed with 30 µl AMPure XP beads (0.6x ratio) as described previously. The resulting library was quantitated on an Agilent TapeStation 4200 System using the High Sensitivity D5000 ScreenTape. Single cell calling card library preparation was performed using the Nextera Mate Pair Sample Prep Kit with modifications to the manufacturer’s protocol. The library was circularized by bringing 300 fmol (approximately 200 ng) of DNA up to a final volume of 268 µl with ddH2O, then adding 30 µl Circularization Buffer 10x and 2 µl Circularization Ligase (final concentration: 1 nM). This reaction was incubated overnight (12-16 hours) at 30ºC. After removal of linear DNA (following manufacturer’s instructions), we sheared the library on a Covaris E220 Focused-ultrasonicator with the following settings–peak power intensity: 200; duty factor: 20%; cycles per burst: 200; time: 40 seconds; temperature: 6ºC. The library preparation was performed per manufacturer’s instructions until adapter ligation. We designed custom adapters (Table S5) so that the standard Illumina sequencing primers would not interfere with our library. Adapters were prepared by combining 4.5 µl of 100 µM scCC_P5_adapter, 4.5 µl of 100 µM scCC_P7_adapter, and 1 µl of NEBuffer 2, then heating in a thermocycler at 95ºC for 5 minutes, then holding at 70ºC for 15 minutes, then ramping down at 1% until it reached 25ºC, holding at that temperature for 5 minutes, before keeping at 4ºC forever. One microliter of this custom adapter mix was used in place of the manufacturer’s recommended DNA Adapter Index. The ligation product was cleaned per manufacturer’s instructions. For the final PCR, the master mix was created by combining 20 µl Enhanced PCR Mix with 28 µl of ddH2O and 1 µl each of 25 µM scCC_P5_primer and 25 µM scCC_P7_primer. This was then added to the streptavidin bead-bound DNA and amplified under the following conditions: 98ºC for 30 seconds; 15 cycles of: 98ºC for 10 seconds–60ºC for 30 seconds–72ºC for 2 minutes; 72ºC for 5 minutes; 4ºC forever. All of the PCR supernatant was transferred to a new tube and purified with 35 µl (0.7x) AMPure XP beads following manufacturer’s instructions. The final library was eluted in 25 µl Elution Buffer and quantitated on an Agilent TapeStation 4200 System using the High Sensitivity D1000 ScreenTape.

treatment_protocol: HCT-116 cells were transfected using Neon electroporation with the aforementioned settings. Each replicate for electroporation was comprised of 2e6 cells. For single cell calling cards analysis of SP1 binding in HCT-116 cells, we transfected four replicates each with 5 µg PB-SRT-Puro and 5 µg SP1-HyPBase. These libraries were not mixed. We used the demultiplexed data from the cell line mixing experiment with HyPBase as controls. Cells were cultured independently and mixed immediately prior to generating single cell emulsions. All cells were allowed to recover for 24 hours before undergoing puromycin selection. A negative control replicate, transfected only with PB-SRT-Puro, was treated identically in parallel. Replicates were harvested once the negative control cells had died.

value: colorectal cancer


title: B3_calb_man_4hrs_250ng

value: Donor B


title: HepG2_HyPBase_scCC

data_processing:
Read 1 sequenced the cell barcode and unique molecular index of each self-reporting transcript. Read 2 began with GGTTAA (end of the piggyBac terminal repeat and insertion site motif) before continuing into the genome. Reads containing this exact hexamer were trimmed using cutadapt with the following settings: -g "^GGTTAA" --minimum-length 1 --discard-untrimmed -e 0 --no-indels. Reads passing this filter were then trimmed of any trailing P7 adapter sequence, again using cutadapt and with the following settings: -a "AGAGACTGGCAAGTACACGTCGCACTCACCATGANNNNNNNNNATCTCGTATGCCGTCTTCTGCTTG" --minimum-length 1. Reads passing these filters were aligned using 10x Genomics’ cellranger with the following settings: --expect-cells=6000 --nosecondary --chemistry=SC3Pv2 --localcores=16 --localmem=30. This workflow also managed barcode validation and collapsing of UMIs. Aligned reads were validated by verifying that they mapped adjacent to TTAA tetramers. Reads were then converted to calling card format (.ccf.txt). Finally, to minimize the presence of intermolecular artifacts, we required that each insertion must have been tagged by at least two different UMIs. We used the set of validated cell barcodes from each scRNA-seq library to demultiplex library-specific barcoded insertions from the scCC data. This approach requires no shared cell barcodes between individual scCC (and scRNA-seq) libraries. As a result, we excluded insertions from non-unique cell barcodes, which represented a very small number of total cells lost (< 1% per multiplexed library).
Genome_build: hg38
Supplementary_files_format_and_content: BAM files contain aligned and annotated calling card reads. Barcode files contain validated single cell barcodes. ccf.txt files contain processed calling card data.  describes calling card-specific BAM tags.

treatment_protocol: HepG2 cells were electroporated with the following settings–pulse voltage: 1,200 V; pulse width: 50 ms; pulse number: 1.  Each replicate for electroporation was comprised of 2e6 cells. For single cell calling cards analysis of FOXA2 binding in HepG2 cells, we transfected six replicates each with 5 µg PB-SRT-Puro; three of these replicates were co-transfected with 5 µg HyPBase, while the other three were co-transfected with 5 µg FOXA2-HyPBase. All cells were allowed to recover for 24 hours before undergoing puromycin selection. A negative control replicate, transfected only with PB-SRT-Puro, was treated identically in parallel. Replicates were harvested once the negative control cells had died.

value: hepatocellular carcinoma


title: OCM1A_BAP1-HyPBase_scCC

treatment_protocol: For single cell calling cards analysis of BAP1 binding in OCM-1A cells, we lipofected (as described above) six replicates each with 1.25 µg PB-SRT-Puro; three of these replicates were co-transfected with 1.25 µg HyPBase, while the other three were co-transfected with 1.25 µg BAP1-HyPBase. We used the TransIT-LT1 transfection reagent following manufacturer’s protocol for 6-well plates. A negative control replicate, transfected only with PB-SRT-Puro, was treated identically in parallel. Replicates were harvested once the negative control cells had died.

value: uveal melanoma


title: Lib10AA_casp8_R3

description: Nextseq


title: DNA_v_RNA_DNACC

description: Bulk DNA calling cards library

data_processing:
Reads were demultiplexed by the 3 base-pair barcode TAG followed by the end of the transposon terminal repeat, culminating with the piggyBac insertion site motif TTAA. Reads that had exact matches to these sequences were hard trimmed using cutadapt (Martin, 2011) with the following settings: -g "^TAGTTTACGCAGACTATCTTTCTAGGGTTAA" --minimum-length 1 --discard-untrimmed -e 0 --no-indels. Reads passing this filter were then trimmed of vector sequence along read 2 using cutadapt with the following settings: -g "^ATCACTTAAGCCGGTAC"" --minimum-length 1 --discard-untrimmed -e 0 --no-indels. The remaining reads were aligned to the human genome (build hg38) with NovoAlign and the following settings: -n 40 -o SAM -o SoftClip. Aligned reads were validated by confirming that they mapped adjacent to the insertion site motif. Successful reads were then converted to calling card format (.ccf.txt; see http://wiki.wubrowse.org/Calling_card) using custom programs (available at https://github.com/arnavm/calling_cards) and visualized on the WashU Epigenome Browser v46 (Zhou et al., 2011) (http://epigenomegateway.wustl.edu/legacy/).
Genome_build: hg38
Supplementary_files_format_and_content: Supplementary_files_format_and_content: BAM files contain aligned and annotated calling card reads. ccf.txt files contain processed calling card data.  describes calling card-specific BAM tags.

extract_protocol:
DNA extraction was performed as described previously (Wang et al., 2012a)
Self-ligation of digested genomic DNA and inverse PCR was performed as described previously (Wang et al., 2012a), with the following modification: digestion with MspI was not performed as the SRT construct contained an second MspI cut site near the terminal repeat.

treatment_protocol: Approximately 500,000 HCT-116 cells were plated in a single well of a 6-well plate. Cells were transfected with 2.5 µg of the SP1-PBase plasmid and 2.5 µg of the PB-SRT-Puro plasmid using Lipofectamine 3000 following manufacturer’s instructions. After 24 hours, cells were split and plated 1:10 in each of three 10 cm dishes. Puromycin was then added to a final concentration of 2 µg/ml and colonies were grown under selection for two weeks. We obtained approximately 2,300 colonies. All cells were pooled together and split into two populations. One half was subjected to DNA extraction, self-ligation, and inverse PCR, as described previously (Wang et al., 2012a), with the following modification: digestion with MspI was not performed as the SRT construct contained an second MspI cut site near the terminal repeat. The other half of cells underwent RNA extraction and SRT library preparation.


title: HepG2_FOXA2-HyPBase_scCC


title: Lib10AA_casp7_R3

description: Nextseq


title: HCT-116_K562_HyPBase_scCC

growth_protocol: HCT-116 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) and K562 cells were cultured in RPMI 1640 Medium supplemented, both supplemented with 10% fetal bovine serum (FBS) and 1% of either penicillin-streptomycin or antibiotic-antimycotic. Cells were grown at 37ºC with 5% carbon dioxide (CO2). Media was replenished every 2 days.

treatment_protocol: HCT-116 cells were transfected using Neon electroporation with the aforementioned settings. K562 cells were electroporated with the following settings–pulse voltage: 1,450 V; pulse width: 10 ms; pulse number: 3. Each replicate for electroporation was comprised of 2e6 cells. For the cell line mixing experiment, we transfected four replicates each of HCT-116 and K562 cells with 5 µg PB-SRT-Puro and 5 µg HyPBase. Cells were cultured independently and mixed immediately prior to generating single cell emulsions. All cells were allowed to recover for 24 hours before undergoing puromycin selection. A negative control replicate, transfected only with PB-SRT-Puro, was treated identically in parallel. Replicates were harvested once the negative control cells had died.

value: mixed tissues


title: Lib10AA_casp8_R1

description: Nextseq


title: Lib10AA_casp7_R2

description: Nextseq


title: K562_SP1-HyPBase_scCC

treatment_protocol: K562 cells were transfected using Neon electroporation with the aforementioned settings. Each replicate for electroporation was comprised of 2e6 cells. For single cell calling cards analysis of SP1 binding in K562 cells, we transfected four replicates each with 5 µg PB-SRT-Puro and 5 µg SP1-HyPBase. These libraries were not mixed. We used the demultiplexed data from the cell line mixing experiment with HyPBase as controls. Cells were cultured independently and mixed immediately prior to generating single cell emulsions. All cells were allowed to recover for 24 hours before undergoing puromycin selection. A negative control replicate, transfected only with PB-SRT-Puro, was treated identically in parallel. Replicates were harvested once the negative control cells had died.

value: leukemia


title: OCM1A_HyPBase_scCC


title: Lib10AA_casp7_R1

description: Nextseq


title: Lib_hP_casp2_R3

description: Nextseq


title: HEK_WT_mock_run1


title: Lib10AA_casp6_R3

description: Nextseq


title: HEK_OEnat10thumpd1_mock_rep2_run2


title: Lib_hP_casp2_R2

description: Nextseq


title: HEK_OEnat10thumpd1_NaCNBH3_rep2_run2


title: HEK_WT_deacetylation_run1

value: deacetylation


title: Lib10AA_casp6_R2

description: Nextseq


title: DNA_v_RNA_RNACC

description:
Bulk DNA calling cards library
The two SRRs for this sample are technical replicates.

data_processing:
Reads were demultiplexed by the N7 index sequences added during the final PCR. Read 1 began with the 3 base-pair barcode followed by the end of the transposon terminal repeat, culminating with the insertion site motif (TTAA in the case of piggyBac; TA in the case of Sleeping Beauty) before entering the genome. piggyBac reads were checked for exact matches to the barcode, transposon sequence, and insertion site at the beginning of reads before being hard trimmed using cutadapt (Martin, 2011) with the following settings: -g "^NNNTTTACGCAGACTATCTTTCTAGGGTTAA" --minimum-length 1 --discard-untrimmed -e 0 --no-indels, where NNN is replaced with the primer barcode. Sleeping Beauty libraries were trimmed with the following settings: -g "^NNNTAAGTGTATGTAAACTTCCGACTTCAACTGTA" --minimum-length 1 --discard-untrimmed -e 0 --no-indels. Reads passing this filter were then trimmed of any trailing Nextera adapter sequence, again using cutadapt and the following settings: -a "CTGTCTCTTATACACATCTCCGAGCCCACGAGACTNNNNNNNNNNTCTCGTATGCCGTCTTCTGCTTG" --minimum-length 1. The remaining reads were aligned to the human genome (build hg38) with NovoAlign and the following settings: -n 40 -o SAM -o SoftClip. Aligned reads were validated by confirming that they mapped adjacent to the insertion site motif. Successful reads were then converted to calling card format (.ccf.txt; see http://wiki.wubrowse.org/Calling_card) using custom programs (available at https://github.com/arnavm/calling_cards) and visualized on the WashU Epigenome Browser v46 (Zhou et al., 2011) (http://epigenomegateway.wustl.edu/legacy/).
Genome_build: hg38
Supplementary_files_format_and_content: BAM files contain aligned and annotated calling card reads. ccf.txt files contain processed calling card data.  describes calling card-specific BAM tags.

extract_protocol:
Total RNA was isolated from each replicate using the RNEasy Plus Mini Kit following manufacturer’s instructions. Briefly, cell pellets were resuspended in 600 µl of Buffer RLT Plus with 1% 2-mercaptoethanol. Cells were homogenized by vortexing. DNA was removed by running lysate through gDNA Eliminator spin columns, while RNA was bound by passing the flow-through over RNEasy spin columns. An on-column treatment with DNaseI was also performed. After washing, RNA was eluted in 40 µl RNase-free H2O. RNA was quantitated using the Qubit RNA HS Assay Kit. We performed first strand synthesis on each replicate with Maxima H Minus Reverse Transcriptase. We mixed 2 µg of total RNA with 1 µl 10 mM dNTPs and 1 µl of 50 µM SMART_dT18VN primer (for a complete list of oligonucleotides, see Table S5), brought the total volume up to 14 µl, and incubated it at 65ºC for 5 minutes. After transferring to ice and letting rest for 1 minute, we added 4 µl 5X Maxima RT Buffer, 1 µl RNaseOUT, and 1 µl of 1:1 Maxima H Minus Reverse Transcriptase diluted in 1x RT Buffer (100 U). The solution was mixed by pipetting and incubated at 50ºC for 1 hour followed by heat inactivation at 85ºC for 10 minutes. Finally, we digested with 1 µl RNaseH at 37ºC for 30 minutes. cDNA was stored at -20ºC.
The PCR conditions for amplifying self-reporting transcripts (i.e. transcripts derived from self-reporting transposons) involved mixing 1 µl cDNA template with 12.5 µl Kapa HiFi HotStart ReadyMix, 0.5 µl 25 µM SMART primer, and either 1 µl of 25 µM SRT_PAC_F1 primer (in the case of puromycin selection) or 0.5 µl of 25 µM SRT_tdTomato_F1 primer (in the case of tdTomato screening). The mixture was brought up to 25 µl with ddH2O. Thermocycling parameters were as follows: 95ºC for 3 minutes; 20 cycles of: 98ºC for 20 seconds–65ºC for 30 seconds–72ºC for 5 minutes; 72ºC for 10 minutes; hold at 4ºC forever. As a control, cDNA quality can be assessed with exon-spanning primers for β-actin (see Table S5 for examples of human primers (Raff et al., 1997)) under the same thermocycling settings. PCR products were purified using AMPure XP beads. 12 µl of resuspended beads were added to the 25 µl PCR product and mixed homogenously by pipetting. After a 5-minute incubation at room temperature, the solution was placed on a magnetic rack for 2 minutes. The supernatant was aspirated and discarded. The pellet was washed twice with 200 µl of 70% ethanol (incubated for 30 seconds each time), discarding the supernatant each time. The pellet was left to dry at room temperature for 2 minutes. To elute, we added 20 µl ddH2O to the pellet, resuspended by pipetting, incubated at room temperature for 2 minutes, and placed on a magnetic rack for one minute. Once clear, the solution was transferred to a clean 1.5 ml tube. DNA concentration was measured on the Qubit 3.0 Fluorometer using the dsDNA High Sensitivity Assay Kit. Calling card libraries from bulk RNA were generated using the Nextera XT DNA Library Preparation Kit. One nanogram of PCR product was resuspended in 5 µl ddH2O. To this mixture we added 10 µl Tagment DNA (TD) Buffer and 5 µl Amplicon Tagment Mix (ATM). After pipetting to mix, we incubated the solution in a thermocycler preheated to 55ºC. The tagmentation reaction was halted by adding 5 µl Neutralization Tagment (NT) Buffer and was kept at room temperature for 5 minutes. The final PCR was set up by adding 15 µl Nextera PCR Mix (NPM), 8 µl ddH2O, 1 µl of 10 µM transposon primer (e.g. OM-PB-NNN) and 1 µl Nextera N7 indexed primer. The transposon primer anneals to the end of the transposon terminal repeat–piggyBac, in the case of OM-PB primers, or Sleeping Beauty, in the case of OM-SB primers–and contains a 3 base pair barcode sequence. Every N7 primer contains a unique index sequence that is demultiplexed by the sequencer. Each replicate was assigned a unique combination of barcoded transposon primer and indexed N7 primer, enabling precise identification of each library’s sequencing reads. The final PCR was run under the following conditions: 95ºC for 30 seconds; 13 cycles of: 95ºC for 10 seconds–50ºC for 30 seconds–72ºC for 30 seconds; 72ºC for 5 minutes; hold at 4ºC forever. After PCR, the final library was purified using 30 µl (0.6x) AMPure XP beads, as described above. The library was eluted in 11 µl ddH2O and quantitated on an Agilent TapeStation 4200 System using the High Sensitivity D1000 ScreenTape.


title: HEK_WT_NaCNBH3_run1

value: WT


title: Lib_hP_casp3_R2

description: Nextseq


title: HEK_OEnat10thumpd1_mock_rep1_run1

value: mock


title: Lib10AA_casp6_R1

description: Nextseq


title: HEK_OEnat10thumpd1_NaCNBH3_rep1_run1


title: Lib10AA_casp3_R3

description: Nextseq


title: Lib10AA_casp2_R3

description: Nextseq


title: Lib10AA_casp3_R2

description: Nextseq


title: HEK_WT_NaCNBH3_run2


title: HEK_OEnat10thumpd1_mock_rep1_run2


title: HEK_OEnat10thumpd1_NaCNBH3_rep1_run2


title: Lib10AA_casp2_R2

description: Nextseq


title: 3T3 and GM12878 mix

description:
sciATAC mouseand human cell line
scATAC_GM12878_counts.txt
scATAC_GM12878_peaks.bed
scATAC_3t3_p1_counts.txt
scATAC_3t3_p1_peaks.bed

organism:
Homo sapiens
Mus musculus

source_name: cell line

tag: strain

value: cell line


title: HEK_OEnat10thumpd1_deacetylation_rep1_run2


title: HCT-116_N2a_HyPBase_scCC

description: Single cell calling cards library

data_processing:
Read 1 sequenced the cell barcode and unique molecular index of each self-reporting transcript. Read 2 began with GGTTAA (end of the piggyBac terminal repeat and insertion site motif) before continuing into the genome. Reads containing this exact hexamer were trimmed using cutadapt with the following settings: -g "^GGTTAA" --minimum-length 1 --discard-untrimmed -e 0 --no-indels. Reads passing this filter were then trimmed of any trailing P7 adapter sequence, again using cutadapt and with the following settings: -a "AGAGACTGGCAAGTACACGTCGCACTCACCATGANNNNNNNNNATCTCGTATGCCGTCTTCTGCTTG" --minimum-length 1. Reads passing these filters were aligned using 10x Genomics’ cellranger with the following settings: --expect-cells=6000 --nosecondary --chemistry=SC3Pv2 --localcores=16 --localmem=30. This workflow also managed barcode validation and collapsing of UMIs. Aligned reads were validated by verifying that they mapped adjacent to TTAA tetramers. Reads were then converted to calling card format (.ccf.txt). Finally, to minimize the presence of intermolecular artifacts, we required that each insertion must have been tagged by at least two different UMIs. We used the set of validated cell barcodes from each scRNA-seq library to demultiplex library-specific barcoded insertions from the scCC data. This approach requires no shared cell barcodes between individual scCC (and scRNA-seq) libraries. As a result, we excluded insertions from non-unique cell barcodes, which represented a very small number of total cells lost (< 1% per multiplexed library). For the species mixing experiment, cells were classified as either human or mouse if at least 80% of self-reporting transcripts in that cell mapped to the human or mouse genome, respectively, and as a multiplet. The estimated multiplet rate was calculated by doubling the observed percentage of human-mouse multiplet, to account for human-human and mouse-mouse doublets.
Genome_build: hg38 & mm10
Supplementary_files_format_and_content: BAM files contain aligned and annotated calling card reads. Barcode files contain validated single cell barcodes. ccf.txt files contain processed calling card data.  describes calling card-specific BAM tags.

treatment_protocol: HCT-116 cells were transfected using Neon electroporation with the aforementioned settings. N2a cells were electroporated with the following settings–pulse voltage: 1,050 V; pulse width: 30 ms; pulse number: 2. Each replicate for electroporation was comprised of 2e6 cells. For the species mixing experiment, we transfected one replicate each of HCT-116 and N2a cells with 5 µg PB-SRT-Puro and 5 µg HyPBase. All cells were allowed to recover for 24 hours before undergoing puromycin selection. A negative control replicate, transfected only with PB-SRT-Puro, was treated identically in parallel. Replicates were harvested once the negative control cells had died.

value: mixed species


title: HEK_WT_mock_run2


title: LV_10_mORF4-NBDY_KO_RIBOseq

city: Utrecht

first: Eva

last: van Rooij

state: Utrecht

address: Uppsalalaan 8

institute: Hubrecht Institute

description:
Ribosomal footprints
WT_vs_mORF4-NBDYKO_RIBOseq.txt

data_processing:
Sequenced RNA-seq and Ribo-seq reads were clipped for adaptor sequences.
Additionally, RNA-seq reads were trimmed for the first 11 and the last 9 bp for each pair.
RNA-seq reads were trimmed to 29-mers in order to avoid mapping or quantification bias due to read length or filtering when comparing to Ribo-seq datasets.
Next, RNA-seq and Ribo-seq reads were filtered for mitochondrial RNA, rRNAs, and tRNAs
Finally, all datasets were mapped to the mouse genome using STAR v2.7.1a, allowing 2 mismatches per read.
To quantify gene expression we counted sequenced reads mapping to CDS regions of genes (Ensembl Release 85), using HTSeq-count.
We obtained a list of differentially expressed and translated genes using DESeq2 and we identified translationally regulated genes by using deltaTE, a method standing for changes in Translational Efficiencies
as described by Chothani S, Adami E, Ouyang JF, Viswanathan S, Hubner N, Cook SA, et al. deltaTE: Detection of Translationally Regulated Genes by Integrative Analysis of Ribo-seq and RNA-seq Data. Curr Protoc Mol Biol. John Wiley & Sons, Ltd; 2019;129:e10
Genome_build: GRCm38/mm10
Supplementary_files_format_and_content: For RNAseq, readcounts of genes are present in a matrix format, with rows being genes and columns being individual mice. Reads are normalized according to the DESeq2 method. Last 4 columns contain the average normalized readcount across all samples, log2 fold change, Pvalue and adjusted Pvalue respectively.
Supplementary_files_format_and_content: For RIBOseq, ribosomal footprint counts of genes are present in a matrix format, with rows being genes and columns being individual mice. Ribosomal footprints are normalized according to the DESeq2 method. Last 4 columns contain the average normalized footprints across all samples, log2 fold change, Pvalue and adjusted Pvalue respectively.
Supplementary_files_format_and_content: For translation efficiency (TE), the efficiency of translation for genes are present in a matrix format, with rows being genes and columns being individual mice. Last 4 columns contain the average translation efficience over all samples, log2 fold change, Pvalue and adjusted Pvalue respectively.

source_name: LV 10 mORF4-NBDY KO

growth_protocol: Mouse were kept unde normal housing conditions

extract_protocol:
RNA was collected using TRIzol reagent according to manufacturer's protocol. Ribosomal footprints were obtaining using the method described by van Heesch S, Witte F, Schneider-Lunitz V, Schulz JF, Adami E, Faber AB, et al. The Translational Landscape of the Human Heart. Cell. United States; 2019;178:242-260.e29.
Illumina mRNA sequencing libraries were prepared using Illumina TruSeq RNA Library Prep Kit v2. Ribosomal footpringing libraries for RIBOseq were prepared using NEXTflex Small RNA-Seq Kit v3.
RNA-seq and RIBOseq

treatment_protocol: No treatments was given to the mice

value: Cardiac (left ventricle)

tag: age

value: 20 weeks

tag: Sex

value: Male


title: Lib10AA_casp3_R1

description: Nextseq

value: phage_PCR and gel extraction


title: HeLa_WT_polyA_mock_rep2

tag: cell line

value: Hela

tag: treatment

value: 37 degrees


title: HEK_WT_deacetylation_run2


title: LV_9_mORF4-NBDY_KO_RIBOseq

source_name: LV 9 mORF4-NBDY KO

tag: tissue


title: Lib10AA_casp2_R1

description: Nextseq


title: HeLa_WT_polyA_mock_rep3


title: HeLa_WT_polyA_NaCNBH3_rep1


title: LV_8_mORF4-NBDY_KO_RIBOseq

source_name: LV 8 mORF4-NBDY KO


title: HeLa_WT_polyA_NaCNBH3_rep2


title: LV_9_mORF4-NBDY_KO_RNAseq

description:
Poly-A RNA
WT_vs_mORF4-NBDYKO_RNAseq.txt


title: HeLa_WT_polyA_mock_rep1


title: LV_10_mORF4-NBDY_KO_RNAseq


title: HEK_OEnat10thumpd1_deacetylation_rep1_run1

value: Nat10/Thumpd1 overexpression


title: LV_8_mORF4-NBDY_KO_RNAseq


title: LV_7_mORF4-NBDY_KO_RIBOseq

source_name: LV 7 mORF4-NBDY KO


title: HeLa_Nat10KD_polyA_NaCNBH3_rep2

value: Nat10 KD


title: LV_7_mORF4-NBDY_KO_RNAseq


title: LV_6_mORF4-NBDY_KO_RIBOseq

source_name: LV 6 mORF4-NBDY KO


title: HeLa_Nat10KD_polyA_NaCNBH3_rep1


title: HeLa_WT_total_mock_rep3


title: HeLa_WT_total_NaCNBH3_rep3


title: LV_4_WT_RIBOseq

source_name: LV 4 WT


title: LV_6_mORF4-NBDY_KO_RNAseq


title: HeLa_Nat10KD_total_NaCNBH3_rep2


title: HeLa_Nat10KD_total_NaCNBH3_rep3


title: HeLa_WT_total_deacetylation_rep3


title: HeLa_WT_polyA_NaCNBH3_rep3


title: LV_3_WT_RNAseq

source_name: LV 3 WT


title: HeLa_WT_total_NaCNBH3_rep2


title: HeLa_WT_total_deacetylation_rep2


title: LV_4_WT_RNAseq


title: LV_1_WT_RIBOseq

source_name: LV 1 WT


title: HeLa_Nat10KD_total_NaCNBH3_rep1


title: LV_2_WT_RNAseq

source_name: LV 2 WT


title: HeLa_WT_total_deacetylation_rep1


title: HeLa_WT_total_mock_rep1


title: LV_2_WT_RIBOseq


title: HeLa_WT_total_NaCNBH3_rep1


title: HeLa_WT_total_mock_rep2


title: HCT116_singlecell_EpCAMlow

city: Rotterdam

first: Mathijs

last: Verhagen

address: Doctor Molewaterplein 40

institute: Erasmus Medical Center

description: single cell

data_processing:
Gene-cell matrices were obtained by conversion of the raw data using the Cell Ranger pipeline (v2.1.1).
Filtered gene-cell matrices were merged in R, and processed for downstream analysis using the Seurat package (v3).
Genome_build: GRCh38
Supplementary_files_format_and_content: Gene-cell matrices include hgnc symbols and read counts

source_name: colon cancer cell line

growth_protocol: Cell populations were sorted from established colon cancer cell lines grown in standard culture medium.

extract_protocol:
Cells were lysed in droplets using Single Cell 3' v2 chemistry (10X Genomics).
RNA-Seq was performed at the Erasmus MC Center for Biomics (Rotterdam, The Netherlands). For each sample, between 50k-100k EpCAMlo cells and EpCAMhi cells were FACS sorted and processed using the 10X Genomics Chromium Single Cell Controller. Samples were sequenced (Illumina) to a depth ranging 49k-65k reads/cells.
single cell RNA-Seq

value: colon

tag: rna isolation date


title: SW480_singlecell_EpCAMlow

description: single cell


title: SW480_singlecell_EpCAMhigh

description: single cell


title: LV_3_WT_RIBOseq


title: HCT116_singlecell_EpCAMhigh

description: single cell


title: LV_1_WT_RNAseq


title: SW480_EpCAMlow_2

data_processing:
IIllumina paired-end reads of 76 bases were trimmed by removing the TrueSeq adapter sequences using Trimmomatic (v.0.33)
Subsequently, the reads were mapped in a two-pass procedure to the human reference genome build hg38 with the RNA-seq aligner STAR (v2.4.2a) and the Homo sapiens GENCODE v23 annotation.
Raw counts were summed with the summarize overlaps function with union mode from the Bioconductor Genomic Alignments package (v1.14.0).
Supplementary_files_format_and_content: tab-delimited text files include Ensemble Gene Id  and read count

growth_protocol: Cell populations were sorted from established colon cancer cell lines grown in standard culture medium. Sorted cells were put in culture for 4 days before RNA was extracted.

extract_protocol:
RNA was harvested using Trizol reagent.
RNA-Seq was performed at the Erasmus MC Center for Biomics (Rotterdam, The Netherlands) according to manufacturer’s instructions (Illumina) and using the TruSeq RNA sample prep kit v2. Briefly, polyA containing mRNA molecules were purified using poly-T oligo attached magnetic beads. Following purification, the mRNA was fragmented into ~200 bp fragments using divalent cations under elevated temperature. The cleaved RNA fragments were copied into first strand cDNA using reverse transcriptase and random primers. This was followed by second strand synthesis using DNA polymerase I and RNaseH treatment. These cDNA fragments were end repaired, a single A base was added and Illumina adapters were ligated. The products were purified and size selected on gel and enriched by PCR. The PCR products were purified by Qiaquick PCR purification and used for cluster generation according to the Illumina cluster generation protocols (www.illumina.com).

treatment_protocol: no treatment was applied


title: SW480_EpCAMlow_3


title: SW480_EpCAMhigh_4


title: SW480_EpCAMlow_1


title: SW480_EpCAMlow_4


title: SW480_EpCAMhigh_2


title: HCT116_EpCAMlow_4


title: SW480_EpCAMhigh_1


title: HCT116_EpCAMlow_3


title: SKIN-SC-010

type: RNA

city: Bethesda

first: Adriana

middle: R

last: Marques

institute: NIAID/NIH

hyb_protocol: Hybridization to the GeneChip Human Genome U133A 2.0 or U133Plus 2.0 Array (Affymetrix, St. Clara, CA) performed according to the manufacturer’s protocol.

scan_protocol: Microarrays were scanned at 570 nm using Affymetrix Genechip Scanner 3000 7G.

data_processing: CEL files were analyzed using Affymetrix Expression Console and the MAS5 algorithm. Resulting Signal Intensity values and Present-Absent calls for each of the probe- sets were processed in JMP statistical package (SAS, Cary, NC) using the Mathematical and Statistical Computing Laboratory (MSCL) Analyst’s Toolbox, written by and freely available (http://abs.cit.nih.gov/MSCLtoolbox/) from one of the authors (P.J.M.). The 22 277 probe sets in common between HG-U133A 2.0 and HG-U133 plus 2.0 chips were used for further analysis. The quantile-normalizing, variance-stabilizing “S10” transform was applied to the Signal Intensity data

label: biotin

source_name: skin- surgical sample

label_protocol: Complementary RNA (cRNA) synthesis and labeling performed according to the manufacturer’s protocol.

growth_protocol: not applicable

extract_protocol: Total RNA was extracted from skin biopsies using a modified RNeasy Fibrous Tissue Mini kit (QIAGEN, Germantown, MD).

treatment_protocol: All skin specimens were stored at −80°C in RNAlater solution (Ambion) before RNA isolation.

value: skin

tag: type

value: skin control- surgical

tag: batch


title: SKIN-SC-015


title: SKIN-SC-016


title: SKIN-SC-012


title: SKIN-SC-013


title: SKIN-SC-006


title: SKIN-SC-014


title: SKIN-SC-008


title: SKIN-SC-004


title: SKIN-SC-003


title: SKIN-SC-005


title: SKIN-SC-011


title: SKIN-SC-007


title: SKIN-SC-001


title: SKIN-SC-002


title: SKIN-SC-009


title: HCT116_EpCAMhigh_3


title: SW480_EpCAMhigh_3


title: HCT116_EpCAMhigh_4


title: HCT116_EpCAMlow_2


title: HCT116_EpCAMhigh_1


title: Human whole brain 2

city: La Jolla

first: Benjamin

last: Gastfriend

address: 9500 Gilman Dr

institute: University of California, San Diego

data_processing:
Basecalling was performed by the Illumina HiSeq 2000
FASTQ files were aligned to the mouse (mm10) or human (hg38) genomes using STAR (v 2.6.0b-1) implemented in Galaxy on the public server at usegalaxy.org
Gene-level transcript abundances were calculated using featureCounts (v 1.6.3) implemented in Galaxy on the public server at usegalaxy.org
Normalized transcript abundances (FPKM, TPM) were calculated from raw counts and featureLengths file (output from featureCounts) using Excel
Genome_build: mm10, hg38
Supplementary_files_format_and_content: Raw counts, FPKM values, and TPM values are provided in an Excel file (xlsx)

source_name: Brain

extract_protocol:
Tissue was lysed with buffer RLT (Qiagen) supplemented with 1% beta-mercaptoethanol. The RNeasy Micro Kit (Qiagen) was used to extract total RNA per manufacturer instructions. DNase I treatment was used to remove gDNA.
Total RNA was subjected to rRNA-depletion and libaries prepared using the NuGEN Ovation Single Cell RNA-Seq High Volume Beta Kit according to manufacturer instructions.

value: whole brain


title: Human whole brain 3


title: HCT116_EpCAMhigh_2


title: SKIN-EM-015

source_name: skin- erythema migrans

value: erythema migrans


title: Human whole brain 1


title: SKIN-EM-012


title: SKIN-EM-010


title: Human brain microvessels 2

value: brain


title: SKIN-EM-011


title: SKIN-EM-013


title: SKIN-EM-014


title: H3K27ac_SU-DHL-4-2

data_processing:
Illumina TruSeq adaptor sequences were trimmed using cutadapt in paired-end mode (non-default parameters: -q 15 -O 4 -m 20)
Reads were aligned to hg19 using bowtie (version 0.12.8; non-default parameters: -m 1 --fr --chunkmbs 500 -n 2 -l 50 --mapq 40 –best) after which duplicate reads were removed using Picard MarkDuplicates
peaks were called using MACS28 (non-default parameters: -q 0.01). Peaks across all samples were combined and overlapping peaks were merged to form a single interval spanning all overlapping peaks. Peaks seen in <2 samples and peaks overlapping ENCODE blacklisted regions [https://sites.google.com/site/anshulkundaje/projects/blacklists]  were removed from further consideration
Genome_build: hg19

source_name: B lymphocyte; metastatic to peritoneal effusion

extract_protocol:
Nuclear lysates were sonicated using a Branson 250 Sonifier (power setting 2, 100% duty cycle for 7 x 30-s intervals). Clarified lysates corresponding to 20 million cells were treated with 1-5ug of antibody against H3K27ac [Abcam #4729]) coupled to Protein G Dynabeads (Life Technologies #10003D, New York). The protein-DNA complexes were washed with RIPA buffer and eluted in 1% SDS TE at 65 °C
ChIP DNA sequencing libraries were generated according to Illumina DNA  TruSeq DNA Sample Preparation Kit Instructions (Illumina Part # FC-121-2001, San Diego,  CA).

treatment_protocol: Cells were crosslinked with formaldehyde at a final concentration of 1% for 10 minutes at room temperature. The reaction was quenched with Glycine at a final concentration of 125mM

tag: disease

value: large cell lymphoma

tag: germ layer

value: Mesoderm


title: H3K27ac_SU-DHL-2-1

source_name: Lymph node; metastatic to peritoneal effusion

value: large cell lymphoma; histiocytic


title: SKIN-EM-005


title: Human brain microvessels 1


title: H3K27ac_SU-DHL-4-1


title: H3K27ac_NCI-H1437-2

source_name: lung; metastatic site to pleural effusion

value: adenocarcinoma; non-small cell lung cancer

value: Endoderm


title: SKIN-EM-002


title: cpc-1KO-0.1%glu_1

city: Dallas

first: xueliang

last: lyu

state: Texas

address: 6001 Forest Park Road

department: Physiology

institute: UT Southwestern Medical Center

data_processing:
Sequencing reads were mapped to reference sequences using tophat2 with parameters --min-intron-length 4 --max-intron-length 5000 --library-type fr-firststrand --max-multihits 20 --read-mismatches 3 --read-gap-length 3 --read-edit-dist 3 --mate-inner-dist 0 --mate-std-dev 50 --no-coverage-search.
Reads Per Kilobase Million (RPKM) were calculated using cufflinks with parameters --min-intron-length 4 --max-intron-length 5000 --library-type fr-firststrand --multi-read-correct --upper-quartile-norm --frag-bias-correct
Genome_build: The reference sequence is whole genome sequence (supercontigs) with gff annotation file from NCBI database (assembly NC12).
Supplementary_files_format_and_content: Tab-delimited text files include RPKM values for each sample.

organism: Neurospora crassa

source_name: mycelia

growth_protocol: Fresh conidia (one week post inoculation on slants) of the WT strain and the cpc-1 deletion mutant were cultured in 2% glucose medium (1 × Vogel’s, 2% glucose) with or without 3-AT, or 0.1% glucose medium (1 × Vogel’s, 0.1% glucose and 0.17% arginine) at room temperate under constant light.

extract_protocol:
Total RNA was extracted using Trizol reagents (Invitrogen) and treated with DNase (Turbo DNase, Ambion) accoding to standard RNA extraction protocal.
Libraries were constucted using Illumina TruSeq reagents. All RNA libraries were prepared for sequencing using standard Illumina protocols.

treatment_protocol: For the samples cultured in 2% glucose medium or 0.1% glucose medium, no special treatment was performed. For the samples treated with 3-AT, the cultures were treated with 5 mM 3-AT for 8 h before sample collection.

value: cpc-1 deletion mutant (cpc-1delta)

value: mycelia

tag: culture conditions


title: cpc-1KO-2%glu_2


title: H3K27ac_SU-DHL-2-2


title: SKIN-EM-008


title: cpc-1KO-0.1%glu_2


title: cpc-1KO-2%glu_1


title: SKIN-EM-009


title: SKIN-EM-007


title: H3K27ac_NP-1

source_name: Neural progenitor

value: normal

value: Ectoderm


title: H3K27ac_NP-2


title: 4200-0.1%glu_1

value: wild-type (WT) strain FGSC4200


title: SKIN-EM-003


title: H3K27ac_NCI-H1437-1


title: 4200-0.1%glu_2


title: SKIN-EM-004


title: H3K27ac_MSLCL-1

source_name: B cell; lymphoblastoid

value: Immortalized with EBV


title: H3K27ac_MSLCL-2


title: SKIN-EM-006


title: H3K27ac_MSIPS-2

source_name: iPS Line

value: Primitive


title: A673cells_siE-EFtreated_rep1

city: Tucson

first: Jacob

middle: C.

last: Schwartz

state: AZ

address: 1501 N Campbell Ave, LSN649

department: Pharmacology

institute: University of Arizona

description:
PolyA-mRNA
siRNA "siE-EF", 5' GACCGCCUAUGCAACUUCUUU 3'

data_processing:
Alignments made to hg38 RefSeq genes downloaded from UCSC genome browser.
Data was aligned using Tophat version 2.1.1.
FPKM values were called using Cufflinks version 2.1.1.
Genome_build: hg38 (GRCh38)
Supplementary_files_format_and_content: FPKM tracking files output by Cuffdiff for each replicate.

extract_protocol:
A673 cells were transfected with 50 nM siRNA as described above. Cells were collected 72 hours post-transfection and total RNA was extracted using TRIzol reagent (ThermoFisher, cat. no. 15596026).
RNA (1 mg) was prepared for sequencing using NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs, cat. no. E7490) to generate sequencing libraries according to the manufacturer’s instructions.

treatment_protocol: A673 cells were reverse transfected with siRNA at 5.0 × 10^5 cells in 6-well dishes using LipofectamineTM RNAiMAX (Invitrogen, cat. no.13778075) according to manufacturer’s instructions. All siRNA transfections were at a final concentration of 50 nM and cells were harvested 72 hours post-transfection for analysis by Western blot.

tag: cell origin

value: Ewing sarcoma

tag: sirna

value: siE-EF

tag: time point

value: 72 hours post-siRNA transfection


title: H3K27ac_MSFIB-2

source_name: skin; fibroblast


title: H3K27ac_MSIPS-1


title: H3K27ac_KU19-1

source_name: urinary bladder transitional cell

value: carcinoma


title: Human brain microvessels 3

value: brain


title: A673cells_siEWSR1treated_rep2

description:
PolyA-mRNA
siRNA "siEWSR1", 5' CUACUAGAUGCAGAGACCCUU 3'


title: A673cells_siEFtreated_rep2

description:
PolyA-mRNA
siRNA "siEF", 5' GCUUCUAGUAGUAGCUGCCUU 3'

value: siEF


title: SKIN-EM-001

value: skin

value: erythema migrans


title: H3K27ac_MSFIB-1

value: Mesoderm


title: H3K27ac_LX-1

source_name: Liver cell

value: Endoderm


title: A673cells_SCRtreated_rep2

description:
PolyA-mRNA
siRNA "SCR", 5' GAUGCAGACAUUCAGGAUGUU 3'

value: SCR


title: A673cells_siE-EFtreated_rep2


title: A673cells_siEWSR1treated_rep1


title: H3K27ac_KU19-2


title: H3K27ac_K1-1

source_name: Thryoid

value: papillary thyroid carcinoma


title: A673cells_SCRtreated_rep1


title: H3K27ac_K562-2

source_name: bone marrow

value: chronic myelogenous leukemia


title: RT112 sh2 rep3

city: Madrid

first: Jaime

last: Martinez de Villarreal

state: Madrid

address: Melchor Fernández Almagro, 3

department: Molecular Oncology Programme

country: Spain

institute: CNIO

data_processing:
Nextpresso 1.9  (Graña O et al. 2017) was used for downstream RNAseq analysis.
Genome_build: hg19
Supplementary_files_format_and_content: Table of normalized transcript counts (FPKM)

source_name: Bladder cancer cell line

growth_protocol: RT112 bladder cancer cells were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% heat-inactivated FBS (Fetal Bovine Serum) and 1 mM sodium pyruvate.

extract_protocol:
Total RNA was extracted using TRIzol (ThermoFisher, ref. 15596026) and purified with the RNeasy Mini Kit (Qiagen, ref. 74104), according to manufacturer’s instructions.
For library preparation, 1 µg of total RNA per condition, each containing an equal amount of ERCC ExFold RNA Spike-In Mix 2 (Ambion, ref. 4456739), was used. Average sample RNA Integrity Number was 9.4 (range 9.0 - 9.8) when assayed on an Agilent 2100 Bioanalyzer. PolyA+ fraction was purified and randomly fragmented, converted to double stranded cDNA and processed through subsequent enzymatic treatments of end-repair, dA-tailing, and ligation to adapters as in Illumina's "TruSeq Stranded mRNA Sample Preparation Part # 15031047 Rev. D" kit (this kit incorporates dUTP during 2nd strand cDNA synthesis, which implies that only the cDNA strand generated during 1st strand synthesis is eventually sequenced). Adapter-ligated library was completed by PCR with Illumina PE primers. The resulting purified cDNA library was applied to an Illumina flow cell for cluster generation and sequenced on an Illumina HiSeq 2500.

treatment_protocol: Cells were transduced with control or STAG2-targeting shRNAs and placed in serum-free medium to avoid cell cycle-dependent effects.

tag: shRNA


title: RT112 shNT rep3

value: control


title: RT112 sh1 rep3


title: H3K27ac_K1-2


title: H3K27ac_K562-1


title: H3K27ac_LX-2

value: normal


title: A673cells_siEFtreated_rep1


title: RT112 sh2 rep1


title: H3K27ac_Jurkat-1

source_name: T lymphocyte

value: acute T cell leukemia


title: RT112 sh1 rep2


title: H3K27ac_Jurkat-2


title: RT112 sh1 rep1


title: H3K27ac_hTERT-HME1-2

source_name: mammary gland

value: Immortalized with hTERT

value: Ectoderm


title: RT112 shNT rep1


title: H3K27ac_hTERT-HME1-1


title: H3K27ac_HT-1376-1

source_name: urinary bladder


title: HCT-116_SP1-HyPBase_scRNA

description:
Single cell RNA-seq library
SRRs represent biological replicates.

data_processing:
scRNA-seq libraries were sequenced on either Illumina HiSeq 2500 or NovaSeq S1 machines. Reads were analyzed using 10x Genomics’ Cell Ranger with the following settings: --expect-cells=6000 --chemistry=SC3Pv2 --localcores=16 --localmem=30. The digital gene expression matrices from 10x were then further processed with scanpy (Wolf et al., 2018) for identification of highly variable genes, batch correction, dimensionality reduction, and Louvain clustering. Processed scRNA-seq datasets were stored as .loom files (http://loompy.org). We cross-referenced gene expression data with published datasets (Rosenberg et al., 2018; Rouillard et al., 2016; Saunders et al., 2018; Tasic et al., 2018; Zeisel et al., 2018) to assign cell types.
Genome_build: hg38
Supplementary_files_format_and_content: Loom files contain processed scRNA-seq data

extract_protocol:
Single cell RNA-seq libraries were prepared using 10x Genomics’ Chromium Single Cell 3’ Library and Gel Bead Kit. Each replicate was targeted for recovery of 6,000 cells. Library preparation followed a modified version of the manufacturer’s protocol. We prepared the Single Cell Master Mix without RT Primer, replacing it with an equivalent volume of Low TE Buffer. Gel-in-emulsion (GEM) generation and GEM-RT incubation proceeded as instructed. At the end of Post GEM-RT cleanup, we added 36.5 µl Elution Solution I and transferred 36 µl of the eluted sample to a new tube (instead of 35.5 µl and 35 µl, respectively). The eluate was split into two 18 µl aliquots and kept at –20ºC until ready for further processing. One fraction was kept for single cell calling cards library preparation, while the other half was further processed into a single cell RNA-seq library.
We added the RT Primer sequence to the products of the scRNA-seq aliquot. We created an RT master mix by adding 20 µl of Maxima 5X RT Buffer, 20 µl of 20% w/v Ficoll PM-400, 10 µl of 10 mM dNTPs, 2.5 µl RNase Inhibitor and 2.5 µl of 100 µM 10x_TSO. To this solution we added 18 µl of the first RT product and 22 µl of ddH2O. Finally, we added 5 µl Maxima H Minus Reverse Transcriptase, mixed by flicking, and centrifuged briefly. This reaction was incubated at 25ºC for 30 minutes followed by 50ºC for 90 minutes and heat inactivated at 85ºC for 5 minutes. The solution was purified using DynaBeads MyOne Silane following 10x Genomics’ instructions, beginning at “Post GEM-RT Cleanup – Silane DynaBeads” step D. The remainder of the single cell RNA-seq protocol, including purification, amplification, fragmentation, and final library amplification, followed manufacturer’s instructions.

value: colorectal cancer


title: HCT-116_SB100

description:
Bulk DNA calling cards library
SRRs represent biological replicates.

treatment_protocol: We cotransfected 10-12 replicates of HCT-116 cells with 5 µg of PB-SRT-Puro plasmid and 5 µg PBase plasmid via Neon electroporation Each replicate contained 2e6 cells. As a negative control, we transfected one replicate of HCT-116 cells with 5 µg PB-SRT-Puro plasmid only. We used the following settings–pulse voltage: 1,530 V; pulse width: 20 ms; pulse number: 1. We used the same experimental setup for experiments with PB-SRT-Puro and each of SP1-PBase, HyPBase, and SP1-HyPBase plasmids, as well as with SB-SRT-Puro and SB100X plasmids. After transfection, each replicate was plated into a 10 cm dish. Puromycin was added after 24 hours to a final concentration of 2 µg/ml. Cells were grown under selection for one week, by which time almost all negative control transfectants were dead. After 7 days, we dissociated each replicate with trypsin-EDTA and created single cell suspensions in phosphate-buffered saline (PBS) Aliquots of each replicate were cryopreserved in cell culture media supplemented with 5% DMSO. The remaining cells were pelleted by centrifugation at 300g for 5 minutes. Cell pellets were either processed immediately or kept at -80ºC in RNAProtect Cell Reagent.


title: H3K27ac_HT-1376-2


title: RT112 sh2 rep2


title: H3K27ac_HT1197-1


title: HCT-116_SP1-HyPBase

data_processing:
Reads were demultiplexed by the N7 index sequences added during the final PCR. Read 1 began with the 3 base-pair barcode followed by the end of the transposon terminal repeat, culminating with the insertion site motif (TTAA in the case of piggyBac; TA in the case of Sleeping Beauty) before entering the genome. piggyBac reads were checked for exact matches to the barcode, transposon sequence, and insertion site at the beginning of reads before being hard trimmed using cutadapt (Martin, 2011) with the following settings: -g "^NNNTTTACGCAGACTATCTTTCTAGGGTTAA" --minimum-length 1 --discard-untrimmed -e 0 --no-indels, where NNN is replaced with the primer barcode. Sleeping Beauty libraries were trimmed with the following settings: -g "^NNNTAAGTGTATGTAAACTTCCGACTTCAACTGTA" --minimum-length 1 --discard-untrimmed -e 0 --no-indels. Reads passing this filter were then trimmed of any trailing Nextera adapter sequence, again using cutadapt and the following settings: -a "CTGTCTCTTATACACATCTCCGAGCCCACGAGACTNNNNNNNNNNTCTCGTATGCCGTCTTCTGCTTG" --minimum-length 1. The remaining reads were aligned to the human genome (build hg38) with NovoAlign and the following settings: -n 40 -o SAM -o SoftClip. Aligned reads were validated by confirming that they mapped adjacent to the insertion site motif. Successful reads were then converted to calling card format (.ccf.txt; see http://wiki.wubrowse.org/Calling_card) using custom programs (available at https://github.com/arnavm/calling_cards) and visualized on the WashU Epigenome Browser v46 (Zhou et al., 2011) (http://epigenomegateway.wustl.edu/legacy/).
Genome_build: hg38
Supplementary_files_format_and_content: BAM files contain aligned and annotated calling card reads. ccf.txt files contain processed calling card data. Peaks file provides details on regions significantly enriched for calling card insertions.  describes calling card-specific BAM tags.


title: HCT-116_K562_HyPBase_scRNA

value: mixed tissues


title: H3K27ac_HT1197-2

value: carcinoma


title: cpc-1KO-2%glu-3-AT_2

value: cpc-1 deletion mutant (cpc-1delta)

value: mycelia


title: H3K27ac_H1-hESC-2

source_name: Embryonic Stem Cell

value: Primitive


title: H3K27ac_H1-hESC-1


title: H3K27ac_GM12878-2

value: Immortalized with EBV


title: cpc-1KO-2%glu-3-AT_3


title: RT112 shNT rep2


title: H3K27ac_HPAEC-2

source_name: pulmonary artery endothelial cells

value: Mesoderm


title: H3K27ac_HPAEC-1


title: 4200-2%glu_2

value: wild-type (WT) strain FGSC4200


title: 4200-2%glu-3-AT_1


title: H3K27ac_HepG2-2

source_name: liver

value: hepatocellular carcinoma

value: Endoderm


title: H3K27ac_HepG2-1


title: cpc-1KO-2%glu-3-AT_1


title: 4200-2%glu-3-AT_3


title: H3K27ac_H9-hESC-2


title: 4200-2%glu_1


title: 4200-2%glu-3-AT_2


title: H3K27ac_H9-hESC-1