title: RA_Y4

city: Guangzhou

first: Yanjuan

last: Chen

address: Guangzhou

department: Department of Geriatrics and Division of Rheumatology and Research

institute: The Second Clinical Medical College, Jinan University (Shenzhen People's Hospital)

data_processing:
The transcriptome sequencing and analysis were conducted by OE biotech Co., Ltd. (Shanghai, China).
Raw data (raw reads) were processed using Trimmomatic
The reads containing ploy-N and the low-quality reads were removed to obtain the clean reads. Then the clean reads were mapped to reference genome using hisat2
Read counts value of each sample was calculated using bowtie2
FPKM value of each gene was calculated using cufflinks, and the read counts of each gene were obtained by htseq-count. Bioinformatics analyses and plotting were performed in R studio software (version 4.2.1).
Assembly: hg19
Supplementary files format and content: Excel file includes raw counts for each Sample
Supplementary files format and content: Excel file includes RPKM values for each Sample

source_name: Peripheral blood

extract_protocol:
Fresh peripheral blood samples were collected in EDTA-K2 anticoagulant tubes separately, transported in a 4°C incubator, and disposed of within 2 hours. PBMCs were isolated from fresh blood by density gradient centrifugation over HISTOPAQUE-1077 (Sigma-Aldrich), according to the manufacturer’s instructions.
Total RNA was extracted using the mirVana miRNA Isolation Kit (Ambion) following the manufacturer’s protocol. RNA integrity was evaluated using the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). The samples with RNA Integrity Number (RIN) ≥ 7 were subjected to the subsequent analysis. The libraries were constructed using TruSeq Stranded mRNA LTSample Prep Kit (Illumina, San Diego, CA, USA) according to the manufacturer’s instructions.
These libraries were sequenced on the Illumina sequencing platform (HiSeq X Ten) and 125bp/150bp paired-end reads were generated.

value: Peripheral blood

value: Peripheral blood mononuclear cells (PBMCs)

tag: genotype

value: Young RA patient

tag: group

value: Young


title: NKCs, Donor 3 (1G)

status: Public on Aug 22 2024

type: SRA

city: Winston-Salem

first: Kimberly

middle: D

last: Reeves

state: North Carolina

address: Medical Center Blvd

country: USA

institute: Wake Forest School of Medicine

library_source: transcriptomic

data_processing:
Raw sequence reads were imported into Partek Flow (Version: 10.0.23.0720) for analysis.
CUTADAPT was used to trim sequence reads based on quality and to remove sequencing adapters; rDNA and mtrDNA contaminants were filtered out using Bowtie 2.
High-quality reads were mapped to the human GENCODE reference database (release 36) using STAR (Spliced Transcripts Alignment to a Reference).
Aligned reads were quantified using the Partek E/M algorithm and then normalized with the median-of-ratios method used in DESeq2.
Assembly: hg38 GENCODE Genes, release 36
Supplementary files format and content: The 'Normalized_Log2Transformed_Genes.txt' processed data file contains normalized and log2-transformed read counts for genes identified in each of the samples. The first column contains the gene symbol, the second column includes the havana gene ID, the third column includes the hgnc ID, and the subsequent columns contain the normalized counts for each designated gene.
Supplementary files format and content: The 'Normalized_Log2Transformed_Transcripts.txt' processed data file contains normalized and log2-transformed read counts for transcripts identified in each of the samples. The first column contains the gene symbol, second column includes ensembl transcript ID, third column includes ensembl gene ID, fourth column includes havana transcript ID, fifth column includes havana gene ID, sixth column includes hgnc ID, seventh column includes transcript type, and the subsequent columns contain the normalized counts for each designated transcript.

molecule: total RNA

organism: Homo sapiens

source_name: Blood

extract_protocol:
RNA was extracted using a Qiagen RNeasy Kit. The quantity of the RNA extracts was determined using a NanoDrop Spectrophotometer, and the integrity of each extract was determined using an Agilent 4200 TapeStation and reagents.
cDNA libraries were prepared using a Perkin Elmer NEXTFLEX Combo-Seq mRNA/miRNA Kit. Proper sizing of cDNA libraries were validated using an Agilent 4200 TapeStation, and library quantities were determined by qPCR using a KAPA Library Quantification Kit (Roche). The cDNA libraries were then normalized prior to pooling to ensure equal library representation. Pooled cDNA libraries were sequenced using an Illumina NovaSeq 6000 System, generating 76-bp single-end reads.

tag: tissue

value: Blood

tag: individual

value: Donor 3

tag: cell type

value: Natural Killer Cells

tag: treatment

value: Normal Gravity


title: Enriched myeloid cells from mouse 19517, PBS

city: Charlottesville

first: Hui

last: Zong

state: Virginia

address: 7042 Pinn Hall

department: Microbiology, Immunology, and Cancer Biology

institute: University of Virginia

description:
toxo_notoxo_macrophages_fpm.csv
M_19517_S5

data_processing:
All sequence reads files (in fastq format) successfully passed ‘Basic Statistic’ check by FastQC (v0.11.5)
The reads were aligned to the mouse genome assembly (mm10) using STAR (v2.7.9a) and quantified with RSEM (v1.3.0)
Differential gene expression analysis was conducted using the standard DESeq2 (v1.36.0) pipeline to identify differentially expressed genes with a false discovery rate less than 0.05
To correct for possible differences between females and males, sex was included in DESeq2 as a covariance.
Assembly: mm10
Supplementary files format and content: DE_toxo_vs_notoxo_macrophages_plus_sex_covariance.csv contains a table with fold changes and p-values for infected vs. PBS comparison as calculated by DESeq2
Supplementary files format and content: toxo_notoxo_macrophages_fpm.csv containes table with fpm walues for all the genes in all the smaples as calculated by DEseq2

organism: Mus musculus

source_name: Tumor

extract_protocol:
RNA were extracted using the RNeasy micro kit (QIAGEN) according to the manufacturer’s instruction. For each sample, 200 ng of total RNAs was used for the construction of sequencing libraries. RNA quality was assessed by Bioanalyzer (Agilent Technologies) with RNA integrity number (RIN) of ≥ 6.5 were selected for library construction.
Illumina TruSeq Stranded Total RNA Library prep kit (#20020596, Illumina) was used to prepare the libraries per Illumina protocol specifications. Ribozero beads, included in the kit, were used to deplete the rRNA. Concentrations of the libraries were determined using the Qubit Flex (#Q33327, Fisher) and the Qubit 1X dsDNA HS Assay kit (#Q33230, fisher).

value: Tumor

value: Myeloid cells

tag: Sex

value: Female

value: PBS

tag: time

value: 27 days after treatment


title: 6780107_tumor_CD45+_cells_scRNAseq

city: Tarrytown

first: Qingqing

last: Wang

state: NY

address: 777 Old Saw Mill River Road

institute: Regeneron Pharmaceuticals

description: 10x Genomics

library_source: transcriptomic single cell

data_processing:
For scRNAseq, raw fastq files were processed with Cell Ranger Single-Cell Software Suite (10X Genomics, v6.1.2) for sample demultiplexing, alignment,filtering and UMI counting. Specifically, alignment was performed against a custom-built genome reference using cellranger mkref as instructed by 10X Genomics, where the humanized LAG3 and PDCD1 sequences were provided in additional to the default mouse genome. Data from multiple samples were merged and further cleaned up to remove dead cell populations, doublets and small tumor sample contaminations, and clustered, analyzed and visualized following the standard analysis workflow of Seurat v4 (cite). Cell identities were annotated using known cell functional markers.
For V(D)J profiling data, Cell Ranger (v6.1.2) was used to perform sample demultiplexing, de novo assembly of read pairs into contigs, alignment and annotation of contigs against the germline segment V(D)J reference sequences from IMGT, labeling and location of CDR3 regions and grouping of clonotypes. Single cell TCR clonotypes data were further analyzed using scRepertoire (v1.13.0) (cite) for clonal analysis. A TCR clonotype is defined as the combination of V(D)J genes comprising the TCR and the nucleotide sequence of the CDR3 region (gene+nucleotide) for paired TCR alpha and beta chains.
Assembly: Mm10
Supplementary files format and content: genes.tsv file, 10X gene file for raw UMI counts
Supplementary files format and content: matrix.mtx file, 10X format, raw UMI counts
Supplementary files format and content: barcodes.tsv file, 10X barcodes file for raw UMI counts
Supplementary files format and content: Metadata.txt file, phenotypic metadata for cells in dataset
Supplementary files format and content: UMAP_coordinates.txt file, UMAP coordinates of cells in dataset, from unsupervised clustering based on gene expression profiles
Supplementary files format and content: filtered_contig_annoatations.csv file, filtered VDJ contig information analyzed by Cell ranger v.3.0.2

molecule: polyA RNA

source_name: tumor

growth_protocol: For tumor studies, unless otherwise indicated, different mouse strains were implanted with tumor cells resuspended in HBSS (GibcoTM 14025076) by subcutaneous injection on the right flank. The following tumor cell numbers were used: MC38 (3x105 cells), B16F10 (3x105 cells), MCA205 (1x106 cells), TRAMP-C2 (1x106 cells), Colon26 (1x106 cells). When tumors were established, mice were randomized based on tumor size and treated with indicated molecules and doses on the indicated days via intraperitoneal injection. For the ID8-VEGF/huMUC16D studies, human CD3/MUC16 knock-in mice were subcutaneously implanted with 1x107 tumor cells, resuspended in RMPI1640 medium, and treatments were administered via intravenous injection. Tumor growth was monitored over time using caliper measurements and calculated as (length x width2)/2. Mice were euthanized when tumors reached >2000mm3 or became severely ulcerated. For tumor rechallenge experiments, 3x105 MC38 cells or 1x107 ID8-VEGF cells were implanted on the left flanks of corresponding tumor-free mice more than 75 days after they received and rejected their primary tumor challenges.

extract_protocol:
Single cells suspended in PBS with 0.04% BSA were loaded on a Chromium Single Cell Instrument (10x Genomics).
RNA-seq and V(D)J libraries were prepared using Chromium Single Cell 5’ Library, Gel Beads & Multiplex Kit (10X Genomics). After amplification, cDNA was split into RNA-seq and V(D)J library aliquots. To enrich the V(D)J library aliquot for TCR a/b, the cDNA was split into two 20 ng aliquots and amplified in two rounds using primers designed in-house. Specifically, for first round amplification the primers used were MP147 (ACACTCTTTCCCTACACGACGC) for short R1, MP120 (GCAGACAGACTTGTCACTGGA) for human TRAC, and MP121(CTCTGCTTCTGATGGCTCAAACA) for human TRBC. For second round amplification, 20 ng aliquots from the first round were amplified using MP147, MP128 (GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGCAGGGTCAGGGTTCTGGATA) a nested R2 plus human TRAC, and MP129 (GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGCAGGGTCAGGGTTCTGGATA) a nested R2 plus human TRABC. V(D)J libraries were prepared from 25 ng each hTRAC and hTRBC amplified cDNA. Paired-end sequencing was performed on Illumina NextSeq500 for RNA-seq libraries (Read 1 26-bp for UMI and cell barcode, 8-bp i7 sample index, and Read 2 55-bp transcript read) and V(D)J libraries (Read 1 150-bp, 8-bp i7 sample index, and Read 2 150-bp read). For RNA-seq libraries, Cell Ranger Single-Cell Software Suite (10X Genomics, v2.2.0) was used to perform sample demultiplexing, alignment, filtering, and UMI counting. The human GRCh38 genome assembly and RefSeq gene model for human were used for the alignment. For V(D)J libraries, Cell Ranger Single-Cell Software Suite (10x Genomics, v2.2.0) was used to perform sample de-multiplexing, de novo assembly of read pairs into contigs, align and annotate contigs against the germline segment V(D)J reference sequences from IMGT, label and locate CDR3 regions, group clonotypes.
RNA-seq and V(D)J seq

treatment_protocol: Human PD-1 knock-in mice bearing established MC38 tumors (200 mm3) were grouped (4 mice per group) and intraperitoneally injected with two doses of either isotype (10 mg/kg), anti-PD1 (10mg/kg), NT-IL2Ra-IL2 (0.5mg/kg), or PD1-IL2Ra-IL2 (0.5mg/kg) on days 0 and 3. On day 6, tumors were excised and dissociated. Enriched CD45+ immune cells from tumors were subjected to single-cell RNA and TCR sequencing analyses

value: tumor


title: 6780108_tumor_CD45+_cells_scRNAseq

description: 10x Genomics


title: Enriched myeloid cells from mouse 19510, PBS

description:
toxo_notoxo_macrophages_fpm.csv
M_19510_S4


title: RA_Y3

state: Guangdong

country: China

tag: cell line


title: NKCs, Donor 2 (uG)

value: Donor 2

value: Simulated Microgravity


title: 6780103_tumor_CD45+_cells_scRNAseq

description: 10x Genomics


title: 5FC derivative clone I1, bio rep 3

city: Vienna

first: Trinh

last: Phan-Canh

email: phancanht99@univie.ac.at, phancanhtrinh@gmail.com, karl.kuchler@univie.ac.at, karl.kuchler@univie.ac.at

state: Vienna

address: Dr.-Bohr-Gasse 9

department: Max Perutz Labs Vienna, Campus Vienna Biocenter

country: Austria

institute: Medical University of Vienna

data_processing:
The quality of raw reads were analyzed using FastQC (Andrews, 2010).
TrueSeq (Illumina) adapters were trimmed by using cutadapt v2.8 (https://cutadapt.readthedocs.io/en/stable/; settings: -q 30 -O 1). The reads were then mapped to C. auris genome (B8441) using NextGenMap (Sedlazeck et al., 2013), settings: -b -Q 30.
Optical read duplicates were removed using Picard tools (Broad Institute, https://broadinstitute.github.io/picard/, settings: MarkDuplicates REMOVE_DUPLICATES=true VALIDATION_STRINGENCY=LENIENT). Read counting was done using HTseq (Anders et al., 2014) in the union mode and the genomic annotation from C. auris B8441 (settings: -f bam -t gene -i ID).
Assembly: Differential gene expression analysis was done using the glm approach in edgeR (Robinson et al., 2009)
Supplementary files format and content: tab delimited .txt files containing the output of the HTSeq analysis (read counts)
Assembly: Candida auris B8441 - Genome version: s01-m01-r22 from Candida Genome Database
Supplementary files format and content: Matrix files are provided for each sample, comprising two columns: GeneID (Candida Genome Database) and raw read count.

organism: Candidozyma auris

source_name: 1133/P/13R, 5FC-induced mutant

growth_protocol: YPD, 37°C, 200 rpm, exponential phase

extract_protocol:
The total RNA was isolated from frozen samples using TRIZOL reagent.
Messenger RNA was purified from total RNA using poly-T oligo-attached magnetic beads. After fragmentation, the first strand cDNA was synthesized using random hexamer primers followed by the second strand cDNA synthesis. The library was ready after end repair, A-tailing, adapter ligation, size selection, amplification, and purification.

treatment_protocol: comparing different genotypes

value: 1133/P/13R, 5FC-induced mutant

value: planktonic cell at exponential phase

value: 5FC-induced mutant

value: YPD


title: NKCs, Donor 2 (1G)


title: 143B_XD23_48h_a

city: Shenzhen

first: jianhui

last: liang

address: 1068 Xueyuan Avenue, Nanshan District

institute: Shenzhen Institute of Science and Technology

data_processing:
All of the analyses were performed using an in-house pipeline from Shanghai Applied Protein Technology。Raw data (or Raw reads) of fastq format were firstly processed through in-house perl scripts. Inthis step, remove the adapter sequence and filter out low quality (low quality, the number of lines with a string quality value less than or equal to 25 accounts for more than 60% of the entire reading) and N (N means that the base information cannot be determined) ratio is greater than 5% reads to obtain clean reads that can be used for subsequent analysis.Then clean reads were separately aligned to reference genome with orientation mode using HISAT2 software (http://daehwankimlab.github.io/hisat2/) to obtain mapped reads.
FeatureCounts (http://subread.sourceforge.net/) was used to count the reads numbers mapped to each gene. And then FPKM of each gene was calculated based on the length of the gene and reads count mapped to this gene.
Differential expression analysis was performed using the DESeq2 (http://bioconductor.org/packages/release/bioc/html/DESeq2.html), DEGs with | log2FC | > 1 and Padj < 0.05 were considered to be significantly different expressed genes.
The GO and KEGG enrichment analysis of differential genes can explain the functional enrichment of differential genes and clarify the differences between samples at the gene function level. We use clusterProfiler R software package for GO function enrichment and KEGG pathway enrichment analysis. When P<0.05, it is considered that the GO or KEGG function is significantly enriched.
Transcription factor is a kind of proteins that can bind to specific DNA sequences, widely existing in organisms. It can be recognized and bound to the cis-acting elements in the upstream regulatory region of genes through specific functional domains, which can activate or hinder the expression of genes. TF analysis of DEGs are extracted directly from the AnimalTFDB (http://bioinfo.life.hust.edu.cn/AnimalTFDB/) /PlantTFDB database(http://planttfdb.cbi.pku.edu.cn/) .
For the species are not existed in the database,fistly, we annotate the genes in the Pfam database (http://pfam.xfam.org/) by interscan tool to find the genes and the DBD database (https://dblp.uni- trier.de/rec/journals/nar/KummerfeldT06.html) to screen for transcription factors.
PPI analysis is used to study whether there is interaction between gene products -- proteins. The analysis is based on the protein information corresponding to genes. Based on the STRING database (https://www.string-db.org/) , which known and predicted Protein-Protein Interactions. For the species existing in the database, we construct the networks by extract the target gene list from the database. For species not included in the database, the target gene set sequence is first aligned with the reference sample protein sequence contained in the STRING protein interaction database with blastx, and the protein interaction relationship of the reference species is used to establish an interaction network.Alternative Splicing is an important mechanism for regulate the expression of genes and the variable of protein.
rMATS (http://rnaseq-mats.sourceforge.net/index.html) is a variable splicing analysis software suitable for RNA-Seq data. It can not only classify variable splicing events, but also classify deformable splicing events as follows: Skipped exon (SE), Alternative 5 splice site (A5SS), Alternative 3 splice site (A3SS), mutually exclusion exon (MXE) , Retained intron (RI).
Assembly: hg38
Supplementary files format and content: gene_fpkm.txt

growth_protocol: The 143B cell lines were propagated in RPMI-1640 medium enriched with 10% fetal bovine serum (Gibco, US) and 1% penicillin-streptomycin (Gibco, US).

extract_protocol:
The cell samples were homogenized in Trizol (Invitrogen) and processed for RNA extraction according to the manufacturer’s instructions.
Paired-end libraries were prepared using a ABclonal mRNA-seq Lib Prep Kit (ABclonal, China) following the manufacturer’s instructions. The mRNA was purified from 1 μg total RNA using oligo (dT) magnetic beads followed by fragmentation carried out using divalent cations at elevated temperatures in ABclonal First Strand Synthesis Reaction Buffer. Subsequently, first-strand cDNAs were synthesized with random hexamer primers and Reverse Transcriptase (RNase H) using mRNA fragments as templates, followed by second-strand cDNA synthesis using DNA polymerase I, RNAseH, buffer, and dNTPs. The synthesized double stranded cDNA fragments were then adapterligated for preparation of the paired-end library. Adaptor-ligated cDNA were used for PCR amplification. PCR products were purified (AMPure XP system) and library quality was assessed on an Agilent Bioanalyzer 4150 system

treatment_protocol: 143B cells were treated with 0.5μM XD23 for 24h or 48h.

value: human osteosarcoma cell line


title: RA_A5

value: Peripheral blood

value: Peripheral blood mononuclear cells (PBMCs)

value: Aged RA patient

value: Aged


title: 6780091_tumor_CD45+_cells_scRNAseq

description: 10x Genomics


title: Enriched myeloid cells from mouse 19122, PBS

description:
toxo_notoxo_macrophages_fpm.csv
M_19122_S3

value: Tumor


title: RA_Y2

value: Young RA patient

value: Young


title: Enriched myeloid cells from mouse 19184, PBS

description:
toxo_notoxo_macrophages_fpm.csv
M_19184_S1

value: Myeloid cells

value: Female

value: PBS


title: K562-Sniper-Mismatch_Lib2-Rep1

city: Hangzhou

first: Lijia

last: Ma

state: Zhejiang

address: Shilongshan Road No.18, Cloud Town, Xihu District

department: School of Life Sciences

institute: Westlake university

library_source: genomic

data_processing:
Reads adapter were removed by cutadapt, then the reads were mapped to the library using bowtie2.  oligo with synthesis error in the  gRNA region were removed.
The repairing pattern was classified according to the sequecing reads and the library design.
Assembly: custermized oligo library
Library strategy: Amplicon Sequencing

molecule: genomic DNA

growth_protocol: HEK293T cell was grown in high-glucose Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (Gemini #900-108) and 1% penicillin/streptomycin (Gibco #15140-122). K562 and Jurkat cells were grown in RPMI 1640 (SIGMA #R8758) with 10% FBS (Gemini #900-108) and 1% penicillin/streptomycin. K562 and Jurkat cells were harvested at day-3.5 post-transduction.

extract_protocol:
Genomic DNA (gDNA) was isolated using DNeasy Blood & Tissue Kits according to the manufacturer’s protocol (Qiagen).
The region that was integrated with genomic DNA including both the gRNA, target sequence, and barcode was PCR amplified from genomic DNA for HTS. Primers for HTS were used to specifically amplify all of the gDNA using 25 µl NEBNext Ultra II Q5 Master Mix (New England Biolabs), 17 µl of gDNA (~2.5ug), 2.5 µl of primer mix at a final concentration of 250 nM, and 5.5 µl water. For each gDNA sample, PCR reactions were in the range from 30 to 60.

treatment_protocol: For screening in Jurkat-Cas9 and K562-cas9 cell line using “ON” and “OFF” library, 15 million cells were resuspended with 6ml RPMI in the presence of 8 µg/ml polybrene, and we transferred 1ml cell suspension into each well of 6-well plate. Predetermined volume of virus in the same 6-well format was added into each well to achieve a MOI of 5. After centrifugation at 600g for 2h at 32°C, 2ml pre-warmed RPMI with 15% FBS and 8 µg/ml polybrene was immediately added into each well of 6-well plate. Cells were selected with puromycin (2.5-3 µg/ml) for 2d (Jurkat-cas9 and K562-cas9)  to remove uninfected cells. Cells were harvested at day 3.5 after transduction.

value: myelogenousleukemiacell


title: mouse, skeletal muscle, quadriceps, WT, postnatal day 90, rep3

city: Poznan

first: Agnieszka

last: Mykowska

address: Uniwersytetu Poznanskiego 6

country: Poland

institute: Adam Mickiewicz University

data_processing:
Reads with lengths between 10 and 35 nt were selected with cutadapt (v1.18) for downstream analysis.
Raw reads were aligned to the Mus musculus genome (GRCm38 downloaded from the Ensembl database) with Bowtie2 (v2.4.1). The mapped reads were counted with featureCounts (v2.0.1) using the Mus musculus miRNA annotation file from mirBase, release version 22.
Differential expression of miRNAs was analyzed with the DESeq2 (v1.30.1) package using the likelihood ratio test (LRT) in R.
Assembly: Ensembl, GRCm38
Supplementary files format and content: Tab-separated text file containing raw counts for miRNA

source_name: skeletal muscle, quadriceps

extract_protocol:
Total RNA was extracted from skeletal muscles in TRI reagent (Sigma) using a bead-based homogenizer (TissueLyser II, Qiagen) (2x 45 sec, max frequency) and 1.4-mm and 2.8-mm ceramic beads (Qiagen). Rapid RNA purification was performed using a Total RNA Zol-out kit (A&A Biotechnology) including DNase treatment according to the manufacturer’s protocol.
200 ng of total RNA was used to prepare libraries with a NEXTflex Small RNA-Seq Kit (Bioo Scientific)

value: skeletal muscle, quadriceps

tag: age

value: 90 days


title: Jurkat-Sniper-Mismatch_Lib2-Rep1

value: T lymphocyte cells

value: Jurkat-Sniper-Mismatch


title: Jurkat-NG-Mismatch_Lib2-Rep1

value: Jurkat-NG-Mismatch


title: RA_Y1


title: K562-NG-Mismatch_Lib2-Rep1


title: RA_A2


title: K562-LZ-Mismatch_Lib2-Rep1


title: RA_A3


title: Jurkat-LZ-Mismatch_Lib2-Rep1

value: Jurkat-LZ-Mismatch


title: Enriched myeloid cells from mouse 19120, PBS

description:
toxo_notoxo_macrophages_fpm.csv
M_19120_S2

value: 27 days after treatment


title: K562-Hifi-Mismatch_Lib2-Rep1


title: RKO-dox-Cas9, sgERH, IL-1beta, biol rep 2

first: Gijs

middle: A.

last: Versteeg

address: Dr. -Bohrgasse 9

department: Department of Microbiology

institute: Max Perutz Labs, University of Vienna

data_processing:
For RNA-seq analysis, quality control was done with fastqc (v0.11.8) and pre-processing (adapter and quality trimming) with trim_galore (v0.6.2) . Cell-line specific 3’-ends were determined with 3-GAmES (https://github.com/poojabhat1690/3-GAmES) using human (GRCh38) RefSeq and Ensembl annotations . QuantSeq reads were quantified with SlamDunk (v0.3.4, Neumann et al., 2019 ) using the counting Windows output from 3-GAmES and 3’-end read counts were collapsed per gene.
Assembly: Hg38
Supplementary files format and content: tab-delimited .csv files including count and TPM values

source_name: Colon carcinoma

growth_protocol: RKO cells were maintained in RPMI 1640 (Thermo Fisher Scientific, 21875) supplemented with  10% FBS (Sigma-Aldrich, F7524),L-glutamine (4 mM, Sigma-Aldrich, G7513), sodium pyruvate (1 mM, Sigma-Aldrich, S8636),  and 1% penicillin/streptomycin (Sigma-Aldrich, P4333) in a humidified incubator at 37 °C and 5% CO2.

extract_protocol:
Total RNA was isolated using Trizol (Invitrongen, 15596018).
3’ mRNA sequencing libraries were generated according to manufacturer’s recommendations (QuantSeq 3′ mRNA-Seq Library Prep Kit FWD for Illumina and PCR Add-on Kit for Illumina, Lexogen).

treatment_protocol:
Cas9 induced knockouts were generated by 5 days of doxycycline (Sigma-Aldrich,D9891) treatment (100 ng/mL).
Stimulation of immune pathways was done by treating the cells with 25 ng/mL IFNγ (Enzo Life Sciences, ENZ-PRT141-0100), 10 ng/mL IL-1beta (Peprotech, 200-01B-10µG), or non-treated (negative control) for 4 hours.

value: Colon carcinoma

value: RKO

value: sgERH

value: 5 days dox


title: mouse, skeletal muscle, quadriceps, WT, postnatal day 14, rep2

value: 14 days


title: RKO-dox-Cas9, sgERH, IL-1beta, biol rep 1


title: RA_A4

value: Peripheral blood

value: Peripheral blood mononuclear cells (PBMCs)

value: Aged RA patient

value: Aged


title: RA_A1


title: mouse, skeletal muscle, quadriceps, WT, postnatal day 14, rep3


title: K562-Sniper-Mismatch_Lib2-Rep2

value: myelogenousleukemiacell


title: RKO-dox-Cas9, sgAAVS1, IL-1beta, biol rep 1


title: Young Femur All

city: Münster

first: Kai

last: Kruse

state: North Rhine-Westphalia

address: Roentgenstr. 20

department: Bioinformatics Core Facility

country: Germany

institute: Max Planck Institute for Molecular Biomedicine

description: BD Rhapsody

data_processing:
STAR version 2.7.10a was used to generate a reference genome index for GRCm39, with Gencode annotations vM29, subset to lncRNA and protein-coding genes.
FASTQ reads were mapped against the reference genome index using STAR with the settings “--soloType CB_UMI_Complex --soloCellFilter None --outSAMtype BAM SortedByCoordinate --soloFeatures GeneFull_Ex50pAS --soloCBmatchWLtype 1MM  --soloUMIlen 8 --soloCBwhitelist BD_CLS1.txt BD_CLS2.txt BD_CLS3.txt --runRNGseed 1 --soloMultiMappers EM --readFilesCommand zcat --outSAMattributes NH HI AS nM NM MD jM jI MC ch CB UB GX GN sS CR CY UR UY”.
Libraries using standard BD Rhapsody beads were mapped using the adapter parameters “--soloAdapterSequence NNNNNNNNNACTGGCCTGCGANNNNNNNNNGGTAGCGGTGACA --soloCBposition 2_0_2_8 2_21_2_29 3_1_3_9 --soloUMIposition 3_10_3_17”, libraries with BD Rhapsody enhanced beads with --soloAdapterSequence NNNNNNNNNGTGANNNNNNNNNGACA --soloCBposition 2_0_2_8 2_13_2_21 3_1_3_9 --soloUMIposition 3_10_3_17”.
Raw counts were imported as AnnData objects. We removed low complexity barcodes with the knee plot method, and further filtered out cells with a mitochondrial mRNA content, as well as unusually high total and gene counts using manually determined cutoffs for each sample. Doublets were scored with scrublet.
Finally, each sample’s gene expression matrix was normalized using scran (1.22.1) with Leiden clustering input at resolution 0.5.
Assembly: mm39
Supplementary files format and content: Tab-separated values and MatrixMarket files

source_name: Femur bone marrow

extract_protocol:
Mice from each age group were euthanized and skull and femur were harvested. Skulls were chopped with scissors in FACS buffer before crushed with mortar and pestle; femurs were crushed without chopping. BM stromal samples were dissociated with Collagenase I (Gibco, Cat# 17100-017, 2 mg/ml) and Collagenase IV (Gibco, Cat# 17104-019, 2 mg/ml) in PBS for 20 minutes at 37°C with intermittent shaking. Cell suspensions were strained through a 40 mm mesh filter and washed with FACS buffer. Cells were resuspended and incubated with biotinylated rat monoclonal anti-hematopoietic lineage antibody cocktail (Miltenyi-Biotec, Cat# 130-092-613, 1:50 dilution). Cells were washed, resuspended in FACS buffer with mouse monoclonal anti-Biotin MicroBeads (Miltenyi-Biotec, Cat# 130-105-637, 1:50 dilution) and incubated before being loaded into a magnetic-associated cell sorting (MACS) column (Miltenyi-Biotec, Cat# 130-042-201) for Lin-depletion. Lin-negative cells were further incubated with rat monoclonal anti-CD45 MicroBeads (Miltenyi-Biotec, Cat# 130-052-301, 1:50 dilution), rat monoclonal anti-CD117 MicroBeads (Miltenyi-Biotec, Cat# 130-091-224, 1:50 dilution), biotinylated rat monoclonal anti-CD71 (Biolegend, Cat# 113803, 1:100). Cells were washed, resuspended in FACS buffer with mouse monoclonal anti-Biotin MicroBeads (Miltenyi-Biotec, Cat# 130-105-637, 1:50 dilution) and incubated before being loaded into a magnetic-associated cell sorting (MACS) column (Miltenyi-Biotec, Cat# 130-042-201) for further hematopoietic depletion.
Single cell suspensions were processed with BD Rhapsody Express system according to the manufacturer's instructions. Then, single cell whole transcriptome libraries were prepared using BD Rhapsody WTA Reagent kit (BD Biosciences).

value: Femur bone marrow

tag: developmental stage

value: Young

value: HSPCs and stromal cells

tag: strain

value: Female


title: Jurkat-Hifi-Mismatch_Lib2-Rep1

value: T lymphocyte cells

value: Jurkat-Hifi-Mismatch


title: mouse, skeletal muscle, quadriceps, WT, postnatal day 14, rep1

value: skeletal muscle, quadriceps


title: mouse, skeletal muscle, quadriceps, WT, postnatal day 90, rep1

value: 90 days


title: Young Skull All

description: BD Rhapsody

source_name: Skull bone marrow

value: Skull bone marrow


title: RKO-dox-Cas9, sgERH, IL-1beta, biol rep 3

value: Colon carcinoma

value: RKO

value: sgERH


title: RKO-dox-Cas9, sgAAVS1, IL-1beta, biol rep 2

value: 5 days dox


title: RKO-dox-Cas9, sgAAVS1, IL-1beta, biol rep 3


title: mouse, skeletal muscle, quadriceps, WT, postnatal day 5, rep3

value: 5 days


title: RKO-dox-Cas9, sgERH, IFNgamma, biol rep 3

value: IFNgamma


title: Old Skull All Replicate 3

description: BD Rhapsody

value: Old


title: RKO-dox-Cas9, sgERH, IFNgamma, biol rep 2


title: mouse, skeletal muscle, quadriceps, WT, postnatal day 90, rep2


title: mouse, skeletal muscle, quadriceps, WT, postnatal day 5, rep2


title: Old Skull All Replicate 2

description: BD Rhapsody


title: Jurkat-Sniper-Mismatch_Lib2-Rep2

value: Jurkat-Sniper-Mismatch


title: mouse, skeletal muscle, quadriceps, WT, postnatal day 5, rep1


title: Old Femur All Replicate 2

description: BD Rhapsody

value: Femur bone marrow


title: Jurkat-NG-Mismatch_Lib2-Rep2

value: Jurkat-NG-Mismatch


title: K562-NG-Mismatch_Lib2-Rep2

value: myelogenousleukemiacell


title: RKO-dox-Cas9, sgERH, IFNgamma, biol rep 1


title: mouse, skeletal muscle, quadriceps, WT, postnatal day 1, rep3

value: 1 day


title: mouse, skeletal muscle, quadriceps, WT, postnatal day 1, rep2


title: RKO-dox-Cas9, sgAAVS1, IFNgamma, biol rep 3


title: RKO-dox-Cas9, sgAAVS1, IFNgamma, biol rep 2


title: Young Skull Interparietal

description: BD Rhapsody

value: Young

value: HSPCs and stromal cells

value: Female


title: Young Skull Frontal

description: BD Rhapsody


title: mouse, skeletal muscle, Mbnl2KO, rep3

data_processing:
Adapter clipping with an in-house Python script, and redundant reads were removed with a fastx_collapser tool from the FASTX-Toolkit package.
The reads in FASTA format were then mapped against a set of mouse pre-miRNA sequences obtained from the miRBase 21 database using Megablast from the BLAST package, version 2.2.26.
It was required that the reads mapped in a sense orientation with no mismatches over the full read length. Then, using an in-house Python script, miRNA expression levels were calculated in the following way: raw counts of all reads mapped to a pre-miRNA sequence in a region occupied by an annotated mature miRNA +/- 2 nt were summed.
The raw expression values served as input for subsequent differential expression analysis with DESeq2 using default settings with a threshold of an adjusted P value below 0.05.
Assembly: mouse pre-miRNA sequences obtained from the miRBase 21 database
Supplementary files format and content: Tab-separated text file containing raw reads for miRNA

value: skeletal muscle, quadriceps

value: 90 days


title: RKO-dox-Cas9, sgAAVS1, IFNgamma, biol rep 1

value: Colon carcinoma


title: K562-LZ-Mismatch_Lib2-Rep2


title: Young Skull Parietal

description: BD Rhapsody

value: Skull bone marrow


title: Jurkat-LZ-Mismatch_Lib2-Rep2

value: T lymphocyte cells

value: Jurkat-LZ-Mismatch


title: K562-Sniper-Mismatch_Lib1-Rep1


title: mouse, skeletal muscle, Mbnl2KO, rep2


title: mouse, skeletal muscle, quadriceps, WT, postnatal day 1, rep1


title: RKO-dox-Cas9, sgERH, non-treated, biol rep 3

value: RKO

value: sgERH

value: non-treated

value: 5 days dox


title: Jurkat-Hifi-Mismatch_Lib2-Rep2

value: Jurkat-Hifi-Mismatch


title: RKO-dox-Cas9, sgERH, non-treated, biol rep 1


title: mouse, skeletal muscle, Mbnl2KO, rep1


title: Jurkat-Sniper-Mismatch_Lib1-Rep1

value: Jurkat-Sniper-Mismatch


title: mouse, skeletal muscle, Mbnl1KO, rep3


title: RKO-dox-Cas9, sgERH, non-treated, biol rep 2


title: K562-NG-Mismatch_Lib1-Rep1

value: myelogenousleukemiacell


title: Young Skull HSPC

description: BD Rhapsody


title: RKO-dox-Cas9, sgAAVS1, non-treated, biol rep 3


title: RKO-dox-Cas9, sgAAVS1, non-treated, biol rep 2


title: mouse, skeletal muscle, Mbnl1KO, rep2


title: Jurkat-NG-Mismatch_Lib1-Rep1

value: Jurkat-NG-Mismatch


title: Old Skull HSPC

description: BD Rhapsody

value: Old


title: Young Femur HSPC

description: BD Rhapsody

value: Femur bone marrow

value: Young

value: HSPCs and stromal cells


title: K562-Hifi-Mismatch_Lib2-Rep2


title: RKO-dox-Cas9, sgAAVS1, non-treated, biol rep 1


title: K562-LZ-Mismatch_Lib1-Rep1


title: Young Femur Stromal

description: BD Rhapsody

value: Female


title: mouse, skeletal muscle, WT, rep3

value: skeletal muscle, quadriceps

value: 90 days


title: Jurkat-LZ-Mismatch_Lib1-Rep1

value: T lymphocyte cells


title: Treated 2

city: Mumbai

first: Pooja

middle: Kamal

last: Melwani

state: Maharashtra

address: Mod Lab, Trombay

department: Radiation Biology & Health Science Division

country: India

institute: Bhabha Atomic Research Centre

data_processing:
Aadaptors reads were processed in paired-end mode of the Trimmomatic 0.38
Sequence reads were mapped to the human reference genome GRCh38.p13 (hg38) using splice aware aligner RNA STAR Galaxy Version 2.7.8a
Gene counts from the aligned BAM files were estimated using featureCount in Galaxy (Galaxy Version 2.0.3+galaxy1)
DESeq2 was run on the counts data of treated versus control samples in Galaxy (Galaxy Version 2.11.40.8+galaxy0)
Assembly: hg38
Supplementary files format and content: Text file includes raw count for each sample

growth_protocol: The human breast adenocarcinoma cell line MCF-7 and monocyte cell line U937 were maintained in DMEM and RPMI respectively in a humidified atmosphere at 37°C in 5% CO2. Both the media were supplemented with 10% FBS and 1% Antibiotic solution

extract_protocol:
Total RNA was isolated using RNeasy mini kits (Qiagen, Hilden, Germany). Libraries were prepared from 1µg RNA samples
Libraries were prepared using Truseq RNA sample prep kit V2 as per the manufacturer’s protocol (Illumina, San Diego, USA)

treatment_protocol:
U937 cells were treated with 50 ng/ml PMA for 72 hours to differentiate them in macrophages. Subsequent to three washes, fresh media was added to cells and macrophage conditioned media was collected 24 hours after
MCF-7 cells were treated with macrophage conditioned medium for 48 hours

value: Breast Adenocarcinoma

value: Macrophage conditioned medium


title: K562-Hifi-Mismatch_Lib1-Rep1


title: mouse, skeletal muscle, WT, rep2


title: Old Skull Stromal

description: BD Rhapsody

value: Skull bone marrow


title: Old Femur Stromal

description: BD Rhapsody


title: mouse, skeletal muscle, WT, rep1


title: Control 1

value: Control


title: Treated 1


title: SBC211

city: New York

first: Sydney

middle: B

last: Blattman

address: 1150 St. Nicholas Ave

institute: Columbia University

description:
CRISPRi; metG* pre-exponential
crRNA

data_processing:
Barcode demultiplexing with custom python script
Remove flanking constant sequences with cutadapt 1.18
Reads aligned with bwa version 0.7.17
Features called with custom python scripts that check for PAM sequence
Assembly: Genbank U00096

organism: Escherichia coli

extract_protocol:
mini-prep
PCR to amplify crRNA sequence and add Illumina adapters (W1423, W1451)

treatment_protocol: after exponential IPTG induction

value: metG-pBbS6C-dCas9 (pLlacO1-dCas9)


title: Jurkat-Hifi-Mismatch_Lib1-Rep1

value: Jurkat-Hifi-Mismatch


title: K562-Sniper-Mismatch_Lib1-Rep2


title: SBC210

description:
CRISPRi; metG* lag outgrowth
crRNA

extract_protocol:
mini-prep
PCR to amplify crRNA sequence and add Illumina adapters (W1421, W1452)

treatment_protocol: 2-day semi-solid outgrowth after overnight (lag)


title: Old Skull All Replicate 1

description: BD Rhapsody


title: non-hematopoietic Dach1OE Bone stromal cells at Postnatal day 30

city: Muenster

first: Hyun-Woo

last: Jeong

state: NRW

department: Dept. Tissue Morphogenesis

institute: Max-Planck-Institute for Molecular Biomedicine

description: BD Rhapsody Express

data_processing:
The FASTQ format of sequencing raw data were processed with BD Rhapsody WTA Analysis pipeline (version 1.11) on SevenBridges Genomics online platform (SevenBridges).
Assembly: mm10
Supplementary files format and content: st files include UMI counts for each cell barcode and genes

source_name: Femur and Tibia

extract_protocol:
The surrounding muscle tissue was removed, and the epiphysis was detached from both the femur and tibia. The bones were then treated with collagenase and crushed to obtain a single-cell suspension. Afterward, the cells were washed, filtered, and resuspended in a blocking solution.  Lineage-negative cells were further enriched using CD45 and CD117 microbeads.
The enriched cells were then processed for scRNA-seq using the BD Rhapsody Express system and the BD Rhapsody WTA Reganet kit. Sequencing was performed on an Illumina NextSeq 500 using a High Output kit (150 cycles).

value: Femur and Tibia

value: Male and Female

value: Bone stromal cells

value: Apj CreERT2 - Dach1OE +/f

value: Postnatal day 30

value: Tamoxifen


title: K562-NG-Mismatch_Lib1-Rep2

value: myelogenousleukemiacell


title: mel#6_scT0_vdj

city: Lausanne

first: Marta

middle: A. S.

last: Perez

address: Amphipôle, Quartier UNIL-Sorge

country: Switzerland

institute: SIB - Swiss Institute of Bioinformatics/ UNIL- University of Lausanne

data_processing:
The scTCR-Seq reads were aligned to the GRCh38 reference genome (refdata-cellranger-vdj-GRCh38-alts-ensembl-4.0.0) and quantified using cellranger vdj (10X Genomics, version 4.0.0 and version 6.0.1).
Assembly: GRCh38
Supplementary files format and content: csv file ("all_contig_annotations.csv" output from Cellranger) containing the mapping of unique cells to TCR

source_name: melanoma

extract_protocol:
Baseline tumors, either frozen pieces were thawed in RPMI + 20% FBS on the day of the assay or fresh tissue was directly chopped upon reception. Tissue was dissociated in RPMI + 2% Gelatin (Sigma-Aldrich) + 200 IU·mL-1 Collagenase I (ThermoFisher Scientific) + 400 IU·mL-1 Collagenase IV (ThermoFisher Scientific) + 5 IU·mL-1 Deoxyribonuclease I (Sigma-Aldrich) + 0.1% RNasin Plus RNase Inhibitor (Promega) for 15-30 min at 37°C. Digested cells were filtered using a 70 μm strainer and resuspended in PBS + 1% Gelatin + 0.1% RNasin. Cells were manually counted then stained with 50 µM·mL-1 of Calcein AM (Thermo Fisher Scientific) and FcR blocked (Miltenyi Biotec) for 15 min at RT. Surface staining with anti-CD45 (BioLegend) was performed for 20 min at 4°C. Cells were resuspended in PBS + 0.04% BSA (Sigma-Aldrich) and DAPI (Invitrogen) staining was performed.
40’000 CD45+ cells were sorted on a MoFlo AstriosEQ (Beckman Coulter) and collected in 0.2mL PCR tubes containing 10 μL of PBS + 0.04% BSA + 0.1% Rnasin. After sorting, cells were manually counted with a hemocytometer and viability was assessed using Trypan blue exclusion.  Ex vivo CD45+ cells from tumors were resuspended at a density of 600-1200 cells µL-1 with a viability of >90% and subjected to a 10X Chromium instrument for single-cell analysis. The standard protocol of 10X Genomics was followed and the reagents for the Chromium Single Cell 5’ Library and V(D)J library (v1.0 or v1.1 Chemistry) were used. 15’000 cells were loaded per sample when possible (or the total number of sorted cells if < 15’000 cells). Using a microfluidic technology, single-cell were captured and lysed, mRNA was reverse transcribed to barcoded cDNA using the provided reagents (10X Genomics). 14 PCR cycles were used to amplify cDNA and the final material was divided into two fractions: first fraction was target-enriched for TCRs and V(D)J library was obtained according to manufacturer protocol (10X Genomics). The second fraction was processed for 5’ gene expression library following the manufacturer’s instruction (10X Genomics). Complementary DNA and library quality were examined on a Fragment Analyzer (Agilent) and quantification was performed with the Qubit HS dsDNA assay kit (Invitrogen). Barcoded V(D)J libraries and GEX libraries were pooled and sequenced by an Illumina HiSeq 4000 sequencer.
scTCR-seq

value: melanoma

tag: patient

value: baseline tumor

value: T cells


title: Old Femur HSPC

description: BD Rhapsody

value: Femur bone marrow

value: Old

value: HSPCs and stromal cells

value: Female


title: Old Femur All Replicate 1

description: BD Rhapsody


title: mel#4_scT0_vdj


title: Jurkat-NG-Mismatch_Lib1-Rep2

value: T lymphocyte cells

value: Jurkat-NG-Mismatch


title: non-hematopoietic Bone stromal cells at Postnatal day 30

description: BD Rhapsody Express

value: Dach1OE +/f


title: Control 2

value: Breast Adenocarcinoma

value: Control


title: SBC209

description:
CRISPRi; metG* lag abx
crRNA

extract_protocol:
mini-prep
PCR to amplify crRNA sequence and add Illumina adapters (W1420, W1451)

treatment_protocol: after 27-hr lag abx; 2-day semi-solid outgrowth

value: metG-pBbS6C-dCas9 (pLlacO1-dCas9)


title: Alen treated 12-week-old Bone endothelial cells replicate2

description: 10x Genomics Chromium

data_processing:
The FASTQ-format sequencing raw data was initially processed using UMI-tools (version 1.1.2).
The data was then aligned to the mouse reference genome (mm10) using STAR (version 2.7.10a)
The aligned data was quantified with Subread featureCounts (version 2.0.3) to generate an expression matrix
Assembly: mm10
Supplementary files format and content: tsv files include UMI counts for each cell barcode and genes

extract_protocol:
To enrich bone endothelial cells, single-cell suspensions were prepared from Cdh5-mT/nG reporter femurs and tibias. Muscle tissue was removed, and the bones were treated with collagenase to obtain a single-cell suspension. After multiple washes and filtration, lineage-depleted cells were sorted using FACS to collect GFP-tomato double-positive cells for scRNA-seq.
For scRNA-seq, the sorted cells were processed using a Chromium controller (10X Genomics) and the Chromium single cells 3' reagent kit. Libraries were evaluated and quantified before sequencing on a NextSeq500 sequencer (Illumina) with a High Output Kit (150 cycles).

value: Femur and Tibia

value: Endothelial cells

value: Alendronate


title: mel#3_scT0_vdj


title: Alen treated 12-week-old Bone endothelial cells replicate1

description: 10x Genomics Chromium


title: mel#2_scT0_vjd


title: SBC212

description:
CRISPRi; metG* after exponential
crRNA

extract_protocol:
mini-prep
PCR to amplify crRNA sequence and add Illumina adapters (W1424, W1452)

treatment_protocol: 4.5-hr liquid outgrowth after dilution of SBC211


title: K562-LZ-Mismatch_Lib1-Rep2

value: myelogenousleukemiacell


title: Jurkat-LZ-Mismatch_Lib1-Rep2

value: Jurkat-LZ-Mismatch


title: mouse, skeletal muscle, Mbnl1KO, rep1

value: skeletal muscle, quadriceps

value: 90 days


title: SBC207

description:
CRISPRi; metG* lag abx
crRNA

extract_protocol:
mini-prep
PCR to amplify crRNA sequence and add Illumina adapters (W1438, W1452)

treatment_protocol: after 4-hr lag abx; 2-day semi-solid outgrowth


title: Jurkat-Sniper-Mismatch_Lib1-Rep2

value: Jurkat-Sniper-Mismatch


title: lung#1_scT0_vdj

source_name: lung

value: lung

value: baseline tumor

value: T cells


title: SBC205

description:
CRISPRi; metG* after overnight
crRNA

extract_protocol:
mini-prep
PCR to amplify crRNA sequence and add Illumina adapters (W1436, W1450)

treatment_protocol: after overnight (with IPTG)


title: PTH treated 12-week-old Bone endothelial cells replicate2

description: 10x Genomics Chromium

value: Female

value: Parathyroid hormone


title: PTH treated 12-week-old Bone endothelial cells replicate1

description: 10x Genomics Chromium


title: K562-Hifi-Mismatch_Lib1-Rep2


title: SBC192

description:
CRISPRi; metG* lag outgrowth
crRNA

extract_protocol:
mini-prep
PCR to amplify crRNA sequence and add Illumina adapters (W1434, W1449)

treatment_protocol: 6-hr liquid outgrowth after overnight (lag)


title: mel#1_scT0_vdj

value: melanoma


title: Jurkat-Hifi-Mismatch_Lib1-Rep2

value: T lymphocyte cells

value: Jurkat-Hifi-Mismatch


title: HeLa cells, ITS O-MAP RIP, Pulldown, Rep3

city: Seattle

first: Evan

middle: Erik

last: Kania

state: WA

address: 1959 NE Pacific Street, HSB K554, Box 357750

department: Pharmacology

institute: University of Washington

data_processing:
Raw RNA-seq FASTA files were aligned to a modified GRCh38 genome assembly (Mars, et al, "A Deconvolution Protocol for ChIP-Seq Reveals Analogous Enhancer Structures on the Mouse and Human Ribosomal RNA Genes."), in which all rDNA repeats are replaced by a single copy of the consensus rDNA locus as an extra chromosome, using HISAT2 version 2.2.1 in the paired-end setting with default parameters.
The resulting SAM files were converted to BAM format and sorted using Samtools version 1.15.1.
Mapped reads were quantified using StringTie version 2.2.1 and the StringTie output was prepared for differential expression analysis using the prepDE.py function. The resulting gene count matrix was used for differential expression analysis using DESeq2 with an FDR cutoff of 0.05.
Assembly: GRCh38
Supplementary files format and content: csv containing read counts for each gene
Library strategy: O-MAP RIP

source_name: HeLa

extract_protocol:
For RNA isolation, pellets were thawed on ice and resuspended by gentle pipetting in 1000 µL ice cold RIPA Buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1% (w/v) SDS, 0.5% (w/v) Sodium Deoxycholate, 1% (v/v) Triton X-100, 5 mM EDTA, 0.5 mM DTT), supplemented with 1x EDTA-Free Halt Protease Inhibitor Cocktail, 0.1 U/µL RNase-OUT, and 10mM Sodium Azide, and rocked end-over-end at 4°C for five minutes. Samples were then sheared using a Branson Digital Sonifier 250 outfitted with a double stepped microtip, at 10–12 Watts for 30 s intervals (0.7 s on; 1.3 s off), with 30 s resting steps between intervals, seven intervals total. Samples were held in ice-cold metal thermal blocks throughout sonication. Lysates were then clarified by centrifugation at 15,000 x g for 10 min at 4°C, moved to fresh tubes and diluted with 1 mL Native Lysis Buffer (NLB: 25 mM Tris-HCl pH 7.5, 150 mM KCl, 0.5% (v/v) NP-40, 5 mM EDTA, 0.5 mM DTT), supplemented with 1X Halt Protease Inhibitor Cocktail, 0.1 U/µL RNase-OUT and 10 mM sodium azide. For each sample, 5% was removed as "input," and to the remainder was added 100 µL of Pierce streptavidin magnetic bead slurry (ThermoFisher Scientific) that had been equilibrated by two washes in 1:1 RIPA:NLB. Samples were incubated for 2 hr at room temperature with end-over-end agitation. Beads were then washed with the following series of buffers (1 mL each, 5 min per wash at room-temperature with end-over-end agitation). All buffers were supplemented with 1x EDTA-Free Halt protease inhibitor cocktail, 0.1 U/µL RNase-OUT, and 0.5 mM DTT, unless otherwise noted: (1) RIPA, supplemented with 10 mM Sodium Azide; (2) RIPA alone (3) High Salt Buffer (1 M KCl, 50 mM Tris-HCl pH 7.5, 5 mM EDTA) (4) Urea Buffer (2 M Urea, 50mM Tris-HCl pH 7.5, 5 mM EDTA) (5) 1:1 RIPA:NLB, without protease inhibitors (6) NLB, without protease inhibitors, (7–8) two washes in TE buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA), without protease inhibitors. RNA was isolated from both input and O-MAP-enriched samples by proteolysis in 100 µL Elution Buffer (2% (v/v) N-lauryl sarcoside, 10 mM EDTA, 5 mM DTT, 200 μg proteinase K, in 1x PBS). Reactions were shaken at 700 rpm in a Mixer HC for 1 hour at 42°C, followed by 1 hour at 60°C. RNA was then extracted once with 25:24:1 phenol:chloroform:isoamyl alcohol, and twice thereafter with 24:1 chloroform:isoamyl alcohol. Reactions were supplemented with 15 µg Glycoblue and NaCl to 300 mM, and ethanol precipitated at –20°C overnight. RNA was harvested by centrifugation at 15,000 x g for 30 minutes at 4°C, washed once with 70% ethanol, and resuspended in 84.75 µL nuclease free water. Contaminating DNA was removed by digestion with 5 U RQ1 RNase-free DNase I in 100 µL of the manufacturer’s supplied buffer (1x final concentration) at 37°C for 30 min, and this reaction was terminated by addition of EDTA to 15 mM, final. RNA was purified by phenol extraction and ethanol precipitation, as described above, and resuspended in 15 µL nuclease free water. For ITS O-MAP samples, ribosomal RNA was depleted by RNase-H digestion, using pools of antisense DNA oligonucleotides that targeted mature rRNAs, but not the pre-rRNA "transcribed spacer" domains. Purified RNA samples were quality assessed using an Agilent 4200 TapeStation with a ‘RNA High Sensitivity’ kit. Samples were quantified on a Nanodrop prior to library preparation and 300ng of RNA per sample was used during library preparation. Libraries were prepared using the NebNext Ultra II Directional RNA Library Prep Kit (NEB) according to manufacturer’s instructions. Each library was given a unique index during synthesis. Library concentration was measured using an NebNext Library Quant Kit for Illumina (NEB). Quality was confirmed using an Agilent 4200 TapeStation with a ‘RNA High Sensitivity’ kit.
Sequencing libraries were prepared from 300 ng RNA using the NEBNext Ultra II Directional RNA Library Prep Kit and NEBNext Multiplex Oligos for Illumina, according to the manufacturer’s instructions. Three biological replicates were used for each experimental condition; each library was given a unique index. Libraries were quantified using the NEBNext Library Quant Kit for Illumina, following the manufacturer's instructions, and the quality of these libraries was confirmed using an Agilent 4200 TapeStation with an "DNA High Sensitivity" kit.

treatment_protocol: Cells were first briefly washed with 1X DPBS and then fixed with 2% PFA in 1X PBS for 10 minutes. Fixation was quenched by two five-minute washes with 250 mM glycine in PBS. Cells were then briefly washed with 1X DPBS and permeabilized with 0.5% (v/v) triton-X 100 in PBS, then washed five minutes with DPBS three times. To inactivate endogenous peroxidases, samples were treated with 0.5% (v/v) H2O2, in 1x PBS, for 10 minutes with gentle rocking, and washed twice with 1X DPBS. Samples were then equilibrated in Formamide Wash Buffer (10% (v/v) deionized formamide; 2x SSC; 0.1% (v/v) Tween-20), for five minutes. This buffer was then aspirated, and each sample was treated with 114 µL of Probe Mix (0.1 µM primary oligo probe pool for ITS in 1x Hybridization Buffer: (10% deionized formamide; 2x SSC; 0.1% (v/v) Tween-20; 10% (w/v) dextran sulfate) and this mix was gently spread over the sample by covering with a clean, 30 mm diameter #1.5 thickness glass cover slip. A Formamide Wash Buffer-soaked kimwipe was placed between the wells to maintain humidity during hybridization. Plates were then sealed with Parafilm and incubated without agitation for 8 hours at 37°C. Following primary hybridization, coverslips were removed and cells were washed three times with pre-warmed 30% Formamide Wash Buffer, 10 minutes per wash, at 37°C with gentle rocking. Each well was treated with 50 µL O-MAP Secondary Probe Mix (100 nM SABER1–HRP oligo, in 30% Formamide Hybridization Buffer), and covered with a clean coverslip, as above. Samples were incubated for 1 hour at 37°C, with gentle rocking. Coverslips were then removed, and samples were washed four times with PBST (0.1% (v/v) Tween-20 in 1x PBS), 15 minutes per wash, with gentle rocking at room temperature. Buffer was aspirated, and in situ biotinylation induced by addition of 1 mL Labeling Solution (0.8 µM biotinyl-tyramide, 1 mM H2O2, 1x PBST), and incubation at room temperature for one minute. Biotinylation was halted by addition of PBSTq (Sodium Azide and Sodium Ascorbate (10 mM each, final) in 1x PBST). Cells were harvested by scraping into PBSTq and pelleted by centrifugation at 3,000 x g for 10 minutes. Buffer was aspirated and cells were flash frozen in liquid nitrogen and stored at –80°C until use.

value: HeLa

value: Human cervical cancer

value: ITS O-MAP

tag: fraction

value: Pulldown


title: 75-week-old Bone endothelial cells replicate2

description: 10x Genomics Chromium

value: Femur and Tibia

value: Endothelial cells


title: K562-Sniper-Lib2-Rep1

value: myelogenousleukemiacell


title: SBC191

description:
CRISPRi; metG* after overnight
crRNA

extract_protocol:
mini-prep
PCR to amplify crRNA sequence and add Illumina adapters (W1434, W1447)

value: metG-pBbS6C-dCas9 (pLlacO1-dCas9)


title: 75-week-old Bone endothelial cells replicate1

description: 10x Genomics Chromium


title: HeLa cells, ITS O-MAP RIP, Pulldown, Rep2


title: HeLa cells, ITS O-MAP RIP, Pulldown, Rep1


title: 12-week-old Bone endothelial cells replicate2

description: 10x Genomics Chromium


title: Jurkat-Sniper-Lib2-Rep1

extract_protocol:
Genomic DNA (gDNA) was isolated using DNeasy Blood & Tissue Kits according to the manufacturer’s protocol (Qiagen).
The region that was integrated with genomic DNA including both the gRNA, target sequence, and barcode was PCR amplified from genomic DNA for HTS. Primers for HTS were used to specifically amplify all of the gDNA using 25 µl NEBNext Ultra II Q5 Master Mix (New England Biolabs), 17 µl of gDNA (~2.5ug), 2.5 µl of primer mix at a final concentration of 250 nM, and 5.5 µl water. For each gDNA sample, PCR reactions were in the range from 30 to 60.

value: Jurkat-Sniper


title: SBC133

description:
CRISPRi; wildtype pre-exponential
crRNA

extract_protocol:
mini-prep
PCR to amplify crRNA sequence and add Illumina adapters (W1436, W1457)

treatment_protocol: after exponential aTc induction

value: wt-pWJ445 (pTet-dCas9)


title: SBC208

description:
CRISPRi; metG* lag abx
crRNA

extract_protocol:
mini-prep
PCR to amplify crRNA sequence and add Illumina adapters (W1412, W1450)

treatment_protocol: after 9.5-hr lag abx; 2-day semi-solid outgrowth


title: K562-NG-Lib2-Rep1


title: HeLa cells, ITS O-MAP RIP, Input, Rep3

value: Input


title: 3-week-old Bone endothelial cells replicate2

description: 10x Genomics Chromium

value: Male and Female


title: 12-week-old Bone endothelial cells replicate1

description: 10x Genomics Chromium

value: Female


title: HeLa cells, ITS O-MAP RIP, Input, Rep2


title: SBC132

description:
CRISPRi; wildtype lag outgrowth
crRNA

extract_protocol:
mini-prep
PCR to amplify crRNA sequence and add Illumina adapters (W1436, W1456)

treatment_protocol: 4-hr liquid outgrowth after overnight (lag)


title: K562-LZ-Lib2-Rep1


title: HeLa cells, ITS O-MAP RIP, Input, Rep1


title: 3-week-old Bone endothelial cells replicate1

description: 10x Genomics Chromium


title: SBC130

description:
CRISPRi; wildtype lag outgrowth
crRNA

extract_protocol:
mini-prep
PCR to amplify crRNA sequence and add Illumina adapters (W1436, W1452)

treatment_protocol: 4-hr semi-solid outgrowth after overnight (lag)


title: Jurkat-LZ-Lib2-Rep1

value: Jurkat-LZ


title: SUIT-2 cells, ITS O-MAP ChIP, Pulldown, Rep2

description:
SUIT2_ITS_OMAP_ChIP_Rep2_L2FC.bw
SUIT2_ITS_OMAP_NADs.bed

data_processing:
Deep sequencing reads were trimmed using TrimGalore! with parameters -q 30 --phred33
Trimmed reads were mapped to a modified GRCh38 genome assembly, (Mars, et al, "A Deconvolution Protocol for ChIP-Seq Reveals Analogous Enhancer Structures on the Mouse and Human Ribosomal RNA Genes.") in which all rDNA repeats are replaced by a single copy of the consensus rDNA locus as an extra chromosome, using Bowtie2 version 2.4.4 with arguments -N 1 -L 25.
Duplicate reads were removed with the Picard MarkDuplicates function before peak calling.
Deduplicated BAM files were sorted and indexed with samtools version 1.15.1.
The deepTools bamcoverage function was used to create coverage bigWig files with the parameters  --ignoreDuplicates -p max --normalizeUsing RPKM.
The deepTools bamcompare function was used to create log2 fold change bigWig files with the parameters --ignoreDuplicates --scaleFactorsMethod None -bs 1 -p max --normalizeUsing RPKM.
Nucleolar associated domains were called by merging peaks from Enriched Domain Detector (EDD) and epic2. EDD was performed using default settings and— because NADs are enriched for highly repetitive sequences like centromeres—an empty BED file for the blacklist region. Epic2 peaks were called with the settings --bin-size 50000 -g 2. Peaks from EDD and epic2 were first concatenated and then merged with the BEDtools merge function, using the default settings.
Assembly: GRCh38
Supplementary files format and content: bigWig, BED (containing coordinates of called nucleolar associated domains)
Library strategy: O-MAP ChIP

extract_protocol:
For DNA isolation, thawed pellets were gently resuspended by pipetting in 1000 µL of CLB (20mM Tris pH 8.0, 85mM KCl, 0.5% NP40) supplemented with 1X Halt protease inhibitor cocktail (ThermoFisher Scientific) and 10 mM NaN3 for 10 minutes. Lysates were then clarified by centrifugation at 3,000 x g for 5 minutes at 4℃. Supernatant was aspirated, and lysates were subjected to another round of CLB lysis, clarification, and supernatant aspiration. Lysates were then lysed by gentle pipeting in 1000 µL of NLB (10mM Tris-HCl pH 7.5, 1% NP40, 0.5% Na Deoxycholate, 0.1% SDS) for 10 minutes. Samples were then sheared using a Branson sonicator at 10-12 Watts for 18 30 s intervals (0.7 s on +1.3 s off), with 30 s resting steps between intervals. Samples were held in ice-cold metal thermal blocks throughout sonication. Lysates were then clarified by centrifugation at 15,000 x g for 10 min at 4℃ and moved to fresh tubes. For each sample, 10% was removed as ‘input’; to the remainder was added 100 µL of streptavidin-coated magnetic bead slurry (ThermoFisher Scientific) that had been equilibrated by two washes in NLB. Samples were incubated for 2 hr at room-temperature with end-over-end agitation. Beads were subsequently washed with the following series of buffers (1 mL each, 5 min per wash, room-temperature, with gentle end-over-end agitation): (1) NLB + 5mM EDTA, 10mM NaN3, 1X Halt protease inhibitor cocktail, 150mM NaCl, (2) NLB + 5mM EDTA, 10mM NaN3, 1X Halt protease inhibitor cocktail, (3+4) 1M KCl, 10mM Tris-HCl pH 7.5, 5mM EDTA, (5+6) 2M Urea, 10mM Tris-HCl pH 7.5, 5mM EDTA, (7) 10mM Tris-HCl, 1% SDS, (8+9) 10mM Tris-HCl pH 7.5, 1mM EDTA. Enriched DNA was released from the beads by proteolysis in 100uL of Elution Buffer (2% N-lauryl sarcoside, 10 mM EDTA, 5 mM DTT, in 1X PBS, supplemented with 200 μg proteinase K (ThermoFisher Scientific)) at 65℃, shaking in a Mixer HC (USA Scientific) 700 rpm for 1 hour. Samples were then placed in a thermocycler overnight at 65℃ for proteinase K treatment. DNA was then extracted using 1:1 phenol:chloroform, and a second chloroform cleanup. Samples were then EtOH precipitated in 300mM NaCl supplemented with glycoblue at -20C overnight. DNA was precipitated by centrifugation at 15,000 x g for 30 minutes at 4℃, washed with 70% EtOH, and resuspended in 20 uL nuclease free water. Each sample then received 1 µL RNaseA (ThermoFisher) and incubated at 37℃ for 1 hour. DNA was then extracted using 1:1 phenol:chloroform with two rounds of chloroform cleanup. Samples were then EtOH precipitated in 300mM NaCl supplemented with glycoblue at -20C overnight, washed in 70% EtOH, and resuspended in 20 µL nuclease free water.
Purified DNA samples were quality assessed using agarose electrophoresis to ensure chromatin sheared size was ~200bp. Samples were quantified on a Nanodrop (ThermoFisher) prior to library preparation and 300ng of DNA per sample was used during library preparation. Libraries were prepared using the NebNext Ultra II DNA Library Prep Kit (NEB) according to manufacturer’s instructions. Each library was given a unique index during synthesis. Library concentration was measured using an NebNext Library Quant Kit for Illumina (NEB). Quality was confirmed using an Agilent 4200 TapeStation with a ‘DNA High Sensitivity’ kit.

value: Human pancreatic adenocarcinoma


title: SBC131

description:
CRISPRi; wildtype lag outgrowth
crRNA

extract_protocol:
mini-prep
PCR to amplify crRNA sequence and add Illumina adapters (W1436, W1455)


title: K562-Hifi-Lib2-Rep1


title: human, DM1, skeletal muscles, rep2

library_source: other

data_processing:
Adapter clipping with an in-house Python script, and redundant reads were removed with a fastx_collapser tool from the FASTX-Toolkit package.
The reads in FASTA format were then mapped against a set of human pre-miRNA sequences obtained from the miRBase 21 database using Megablast from the BLAST package, version 2.2.26.
It was required that the reads mapped in a sense orientation with no mismatches over the full read length. Then, using an in-house Python script, miRNA expression levels were calculated in the following way: raw counts of all reads mapped to a pre-miRNA sequence in a region occupied by an annotated mature miRNA +/- 2 nt were summed.
The raw expression values served as input for subsequent differential expression analysis with DESeq2 using default settings with a threshold of an adjusted P value below 0.05.
Assembly: human pre-miRNA sequences obtained from the miRBase 21 database
Supplementary files format and content: Tab-separated text file containing raw counts for miRNA

molecule: other

source_name: skeletal muscle, biceps branchii

extract_protocol:
Total RNA was extracted from skeletal muscles in TRI reagent (Sigma) using a bead-based homogenizer (TissueLyser II, Qiagen) (2x 45 sec, max frequency) and 1.4-mm and 2.8-mm ceramic beads (Qiagen). Rapid RNA purification was performed using a Total RNA Zol-out kit (A&A Biotechnology) including DNase treatment according to the manufacturer’s protocol.
200 ng of total RNA was used to prepare libraries with a NEXTflex Small RNA-Seq Kit (Bioo Scientific)

value: skeletal muscle, biceps branchii

tag: diagnosis

value: myotonic dystrophy type 2

value: female

value: 44 years


title: SBC134

description:
CRISPRi; wildtype after exponential
crRNA

extract_protocol:
mini-prep
PCR to amplify crRNA sequence and add Illumina adapters (W1436, W1458)

treatment_protocol: 3.5-hr liquid outgrowth after dilution of SBC133


title: SUIT-2 cells, ITS O-MAP ChIP, Pulldown, Rep1

description:
SUIT2_ITS_OMAP_ChIP_Rep1_L2FC.bw
SUIT2_ITS_OMAP_NADs.bed

value: ITS O-MAP


title: human, DM1, skeletal muscles, rep3

value: skeletal muscle, quadriceps

value: myotonic dystrophy type 3

value: male


title: SUIT-2 cells, ITS O-MAP ChIP, Input

description:
SUIT2_ITS_OMAP_ChIP_Rep1_L2FC.bw
SUIT2_ITS_OMAP_ChIP_Rep2_L2FC.bw
SUIT2_ITS_OMAP_NADs.bed


title: SBC128

description:
CRISPRi; wildtype lag abx
crRNA

extract_protocol:
mini-prep
PCR to amplify crRNA sequence and add Illumina adapters (W1434, W1457)


title: SBC126

description:
CRISPRi; wildtype after overnight
crRNA

extract_protocol:
mini-prep
PCR to amplify crRNA sequence and add Illumina adapters (W1434, W1455)

treatment_protocol: after overnight (with aTc)


title: Panc 03.27 cells, ITS O-MAP ChIP, Pulldown, Rep2

description:
Panc37_ITS_OMAP_ChIP_Rep2_L2FC.bw
Panc37_ITS_OMAP_NADs.bed

value: Pulldown


title: K562-Sniper-Lib2-Rep2

treatment_protocol: For screening in Jurkat-Cas9 and K562-cas9 cell line using “ON” and “OFF” library, 15 million cells were resuspended with 6ml RPMI in the presence of 8 µg/ml polybrene, and we transferred 1ml cell suspension into each well of 6-well plate. Predetermined volume of virus in the same 6-well format was added into each well to achieve a MOI of 5. After centrifugation at 600g for 2h at 32°C, 2ml pre-warmed RPMI with 15% FBS and 8 µg/ml polybrene was immediately added into each well of 6-well plate. Cells were selected with puromycin (2.5-3 µg/ml) for 2d (Jurkat-cas9 and K562-cas9)  to remove uninfected cells. Cells were harvested at day 3.5 after transduction.

value: myelogenousleukemiacell


title: Jurkat-NG-Lib2-Rep1

value: T lymphocyte cells

value: Jurkat-NG


title: SBC102

description:
CRISPRi; metG* lag outgrowth
crRNA

extract_protocol:
mini-prep
PCR to amplify crRNA sequence and add Illumina adapters (W1423, W1456)

value: metG-pWJ445 (pTet-dCas9)


title: Panc 03.27 cells, ITS O-MAP ChIP, Pulldown, Rep1

description:
Panc37_ITS_OMAP_ChIP_Rep1_L2FC.bw
Panc37_ITS_OMAP_NADs.bed


title: human, non-DM, skeletal muscles, rep2

value: <30 CTG

value: healthy

value: 52 years


title: human, non-DM, skeletal muscles, rep1

value: 35 years


title: Panc 03.27 cells, ITS O-MAP ChIP, Input

description:
Panc37_ITS_OMAP_ChIP_Rep1_L2FC.bw
Panc37_ITS_OMAP_ChIP_Rep2_L2FC.bw
Panc37_ITS_OMAP_NADs.bed

value: Human pancreatic adenocarcinoma

value: Input


title: HEK293T iCLIP3

city: shanghai

first: ql

last: zheng

address: xiangyin

institute: immunity

data_processing:
After linker sequence removal and quality trimming, sequence mapping was performed using Bowtie with default parameters (build mm9).
Bioinformatic and motif enrichment analysis was performed as described previously(Moore, M.J. et al. Mapping Argonaute and conventional RNA-binding protein interactions with RNA at single-nucleotide resolution using HITS-CLIP and CIMS analysis. Nat Protoc 9, 263-293 (2014).)
Genome_build: hg38
Supplementary_files_format_and_content: xlsx files containing the gene ID, peak number,the chromosomal location of the peaks obtained from the iCLIP experiments

growth_protocol: Cells were grown in RIPM-1640 media supplemented with 10% FBS

extract_protocol:
Cells were UV cross-linked and prepared as described previously (Huppertz, I. et al. iCLIP: protein-RNA interactions at nucleotide resolution. Methods 65, 274-287 (2014)) and immunprecipitation of NSP12 using an anti-Myc antibody obtained from CST (2278).
RNA library constructed as described previously (Huppertz, I. et al. iCLIP: protein-RNA interactions at nucleotide resolution. Methods 65, 274-287 (2014))

treatment_protocol: HEK293T cells were transfected with Control or NSP12 plasmids.

tag: iclip antibody

value: Myc CST(2278)


title: K562-NG-Lib2-Rep2


title: SBC129

description:
CRISPRi; wildtype lag abx
crRNA

extract_protocol:
mini-prep
PCR to amplify crRNA sequence and add Illumina adapters (W1434, W1458)

treatment_protocol: after 2-hr lag abx; 2-day semi-solid outgrowth

value: wt-pWJ445 (pTet-dCas9)


title: 8988T cells, ITS O-MAP ChIP, Pulldown, Rep2

description:
8988T_ITS_OMAP_ChIP_Rep2_L2FC.bw
8988T_ITS_OMAP_NADs.bed

value: ITS O-MAP


title: Jurkat-NG-Lib2-Rep2


title: SBC100

description:
CRISPRi; metG* lag outgrowth
crRNA

extract_protocol:
mini-prep
PCR to amplify crRNA sequence and add Illumina adapters (W1423, W1452)


title: HEK293T iCLIP2


title: SBC99

description:
CRISPRi; metG* lag abx
crRNA

extract_protocol:
mini-prep
PCR to amplify crRNA sequence and add Illumina adapters (W1421, W1456)


title: K562-LZ-Lib2-Rep2


title: SBC98

description:
CRISPRi; metG* lag abx
crRNA

extract_protocol:
mini-prep
PCR to amplify crRNA sequence and add Illumina adapters (W1421, W1455)


title: Jurkat-Hifi-Lib2-Rep1

extract_protocol:
Genomic DNA (gDNA) was isolated using DNeasy Blood & Tissue Kits according to the manufacturer’s protocol (Qiagen).
The region that was integrated with genomic DNA including both the gRNA, target sequence, and barcode was PCR amplified from genomic DNA for HTS. Primers for HTS were used to specifically amplify all of the gDNA using 25 µl NEBNext Ultra II Q5 Master Mix (New England Biolabs), 17 µl of gDNA (~2.5ug), 2.5 µl of primer mix at a final concentration of 250 nM, and 5.5 µl water. For each gDNA sample, PCR reactions were in the range from 30 to 60.

value: Jurkat-Hifi


title: 8988T cells, ITS O-MAP ChIP, Input

description:
8988T_ITS_OMAP_ChIP_Rep1_L2FC.bw
8988T_ITS_OMAP_ChIP_Rep2_L2FC.bw
8988T_ITS_OMAP_NADs.bed


title: Jurkat-LZ-Lib2-Rep2

value: Jurkat-LZ


title: NSP12 2

data_processing:
Library strategy: Ribo-Seq
Illumina Casava1.7 software used for basecalling.
For Ribo-seq analyses, the adaptor sequences were first removed from Illumina sequencing reads. while for Ribo-seq reads, the adapters were trimmed, and the random barcodes were extracted and appended to read names by custom script. To remove potential rRNA contamination, the reads were mapped to rRNA sequences with bowtie program 42, and those not mapped were then mapped to the human genome with gene annotation by using STAR program in global mode
For fragments within range from 28 nt to 30 nt, the 13th base located at the 1st position of codons were treated as canonical ribosome-protected fragments (RPFs) for the translation efficiency analysis.
Genome_build: hg19
Supplementary_files_format_and_content: xlsx files containing gene ID, RPKM values and log2FC for each Sample

extract_protocol:
The DNase-treated Control and NSP12 overexpression HEK293T cells lysates were clarified by centrifugation and subjected to ribosome profiling analyses. The lysates were further layered over 10%–60% sucrose gradients to prepare polysome fractions. The obtained polysomes were treated with RNase I and then subjected to purification of 30 nt ribosome protected fragments using a 15% TBE-Urea gel.
After obtaining ribosome footprints, ribosomal profiling libraries were constructed using a NEBNext Multiple Small RNA Library Prep set for Illumina.

treatment_protocol: HEK293T cells were transfected with Control or NSP12 overexpression plasmid

tag: transfection

value: NSP12 overexpression


title: human, DM1, skeletal muscles, rep1

value: skeletal muscle, biceps branchii

tag: genotype

value: myotonic dystrophy type 1

value: female

value: 50 years


title: SBC96

description:
CRISPRi; metG* after overnight
crRNA

extract_protocol:
mini-prep
PCR to amplify crRNA sequence and add Illumina adapters (W1412, W1455)

value: metG-pWJ445 (pTet-dCas9)


title: NSP12 1


title: Jurkat-Sniper-Lib2-Rep2

value: T lymphocyte cells

value: Jurkat-Sniper


title: K562-Hifi-Lib2-Rep2

value: myelogenousleukemiacell


title: AsPC-1 cells, ITS O-MAP ChIP, Pulldown, Rep2

description:
ASPC1_ITS_OMAP_ChIP_Rep2_L2FC.bw
ASPC1_ITS_OMAP_NADs.bed

value: Pulldown


title: SB806d

description:
PETRI-seq; wildtype prolonged stationary phase (6 days) or standard overnight diluted into fresh media for 1 hour or treated with ampicillin
Barcoded cDNA

data_processing:
Barcode demultiplexing with cutadapt 1.18
UMIs extracted with UMI-tools version 0.5.5
Reads aligned with bwa version 0.7.17
Features called with featureCounts version 1.6.3
Final matrix generated by collapsing reads by UMI with a custom script in python 2.7.15
Assembly: Genbank U00096

extract_protocol:
in situ RT and ligations followed by cell lysis with detergent and proteinase K
Double-stranded cDNA was made by second strand synthesis. Libraries were tagmented and amplified using the Nextera XT kit. rRNA was depleted using NEB Cas9 (M0646) and full probe set

treatment_protocol: after 6-day stationary growth, diluted into fresh media, grown 1 hour (d6_1hr_2_20220422); or diluted into fresh media + ampicillin for 4 hours; or standard overnight, diluted into fresh media, grown 1 hour (d1_1hr_1_20220807, WT_1hr_20220331)

value: wildtype


title: AsPC-1 cells, ITS O-MAP ChIP, Pulldown, Rep1

description:
ASPC1_ITS_OMAP_ChIP_Rep1_L2FC.bw
ASPC1_ITS_OMAP_NADs.bed

value: Human pancreatic adenocarcinoma


title: Jurkat-Hifi-Lib2-Rep2


title: SBC101

description:
CRISPRi; metG* lag outgrowth
crRNA

extract_protocol:
mini-prep
PCR to amplify crRNA sequence and add Illumina adapters (W1423, W1455)

treatment_protocol: 2-day semi-solid outgrowth after overnight (lag)


title: SB805d

extract_protocol:
in situ RT and ligations followed by cell lysis with detergent and proteinase K
Double-stranded cDNA was made by second strand synthesis. Libraries were tagmented and amplified using the Nextera XT kit. rRNA was depleted using GenScript Cas9 and full probe set


title: Control 2

value: Control


title: K562-Sniper-Lib1-Rep1


title: AsPC-1 cells, ITS O-MAP ChIP, Input

description:
ASPC1_ITS_OMAP_ChIP_Rep1_L2FC.bw
ASPC1_ITS_OMAP_ChIP_Rep2_L2FC.bw
ASPC1_ITS_OMAP_NADs.bed

extract_protocol:
For DNA isolation, thawed pellets were gently resuspended by pipetting in 1000 µL of CLB (20mM Tris pH 8.0, 85mM KCl, 0.5% NP40) supplemented with 1X Halt protease inhibitor cocktail (ThermoFisher Scientific) and 10 mM NaN3 for 10 minutes. Lysates were then clarified by centrifugation at 3,000 x g for 5 minutes at 4℃. Supernatant was aspirated, and lysates were subjected to another round of CLB lysis, clarification, and supernatant aspiration. Lysates were then lysed by gentle pipeting in 1000 µL of NLB (10mM Tris-HCl pH 7.5, 1% NP40, 0.5% Na Deoxycholate, 0.1% SDS) for 10 minutes. Samples were then sheared using a Branson sonicator at 10-12 Watts for 18 30 s intervals (0.7 s on +1.3 s off), with 30 s resting steps between intervals. Samples were held in ice-cold metal thermal blocks throughout sonication. Lysates were then clarified by centrifugation at 15,000 x g for 10 min at 4℃ and moved to fresh tubes. For each sample, 10% was removed as ‘input’; to the remainder was added 100 µL of streptavidin-coated magnetic bead slurry (ThermoFisher Scientific) that had been equilibrated by two washes in NLB. Samples were incubated for 2 hr at room-temperature with end-over-end agitation. Beads were subsequently washed with the following series of buffers (1 mL each, 5 min per wash, room-temperature, with gentle end-over-end agitation): (1) NLB + 5mM EDTA, 10mM NaN3, 1X Halt protease inhibitor cocktail, 150mM NaCl, (2) NLB + 5mM EDTA, 10mM NaN3, 1X Halt protease inhibitor cocktail, (3+4) 1M KCl, 10mM Tris-HCl pH 7.5, 5mM EDTA, (5+6) 2M Urea, 10mM Tris-HCl pH 7.5, 5mM EDTA, (7) 10mM Tris-HCl, 1% SDS, (8+9) 10mM Tris-HCl pH 7.5, 1mM EDTA. Enriched DNA was released from the beads by proteolysis in 100uL of Elution Buffer (2% N-lauryl sarcoside, 10 mM EDTA, 5 mM DTT, in 1X PBS, supplemented with 200 μg proteinase K (ThermoFisher Scientific)) at 65℃, shaking in a Mixer HC (USA Scientific) 700 rpm for 1 hour. Samples were then placed in a thermocycler overnight at 65℃ for proteinase K treatment. DNA was then extracted using 1:1 phenol:chloroform, and a second chloroform cleanup. Samples were then EtOH precipitated in 300mM NaCl supplemented with glycoblue at -20C overnight. DNA was precipitated by centrifugation at 15,000 x g for 30 minutes at 4℃, washed with 70% EtOH, and resuspended in 20 uL nuclease free water. Each sample then received 1 µL RNaseA (ThermoFisher) and incubated at 37℃ for 1 hour. DNA was then extracted using 1:1 phenol:chloroform with two rounds of chloroform cleanup. Samples were then EtOH precipitated in 300mM NaCl supplemented with glycoblue at -20C overnight, washed in 70% EtOH, and resuspended in 20 µL nuclease free water.
Purified DNA samples were quality assessed using agarose electrophoresis to ensure chromatin sheared size was ~200bp. Samples were quantified on a Nanodrop (ThermoFisher) prior to library preparation and 300ng of DNA per sample was used during library preparation. Libraries were prepared using the NebNext Ultra II DNA Library Prep Kit (NEB) according to manufacturer’s instructions. Each library was given a unique index during synthesis. Library concentration was measured using an NebNext Library Quant Kit for Illumina (NEB). Quality was confirmed using an Agilent 4200 TapeStation with a ‘DNA High Sensitivity’ kit.

value: Input


title: Control 1


title: SB803d


title: 8988T cells, ITS O-MAP ChIP, Pulldown, Rep1

description:
8988T_ITS_OMAP_ChIP_Rep1_L2FC.bw
8988T_ITS_OMAP_NADs.bed

value: ITS O-MAP


title: HEK293T iCLIP1

value: Myc CST(2278)


title: HeLa cells, ITS O-MAP ChIP, Pulldown, Rep2

description:
HeLa_ITS_OMAP_ChIP_Rep2_L2FC.bw
HeLa_ITS_OMAP_ChIP_NADs.bed

value: HeLa

value: Human cervical cancer


title: HEK293T_NSP12_2

data_processing:
Illumina Casava1.7 software used for basecalling.
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence, then mapped to hg19 whole genome using bowtie v0.12.2 with parameters -q -p 4 -e 100 -y -a -m 10 --best --strata
Reads Per Kilobase of exon per Megabase of library size (RPKM) were calculated using a protocol from Chepelev et al., Nucleic Acids Research, 2009. In short, exons from all isoforms of a gene were merged to create one meta-transcript. The number of reads falling in the exons of this meta-transcript were counted and normalized by the size of the meta-transcript and by the size of the library.
Genome_build: hg19 and Cercopithecus aethiops
Supplementary_files_format_and_content: xlsx files containing include RPKM values for each Sample

extract_protocol:
Total RNA was isolated from the above treated cells using Trizol reagent. Illumina TruSeq RNA Sample Prep Kit (Cat#FC-122-1001) was used with 1 ug of total RNA for the construction of sequencing libraries.
RNA libraries were prepared for sequencing using standard Illumina protocols

treatment_protocol: HEK293T or VERO cells were transfected with Control or NSP12 overexpression plasmid

tag: overexpression

value: NSP12 overexpression


title: Jurkat-Sniper-Lib1-Rep1

data_processing:
Reads adapter were removed by cutadapt, then the reads were mapped to the library using bowtie2.  oligo with synthesis error in the  gRNA region were removed.
The repairing pattern was classified according to the sequecing reads and the library design.
Assembly: custermized oligo library
Library strategy: Amplicon Sequencing


title: HeLa cells, ITS O-MAP ChIP, Pulldown, Rep1

description:
HeLa_ITS_OMAP_ChIP_Rep1_L2FC.bw
HeLa_ITS_OMAP_ChIP_NADs.bed


title: K562-NG-Lib1-Rep1


title: HEK293T_NSP12_1


title: SB796

description:
PETRI-seq; wildtype after standard overnight diluted into fresh media for 1 hour
Barcoded cDNA

extract_protocol:
in situ RT and ligations followed by cell lysis with detergent and proteinase K
Double-stranded cDNA was made by second strand synthesis. Libraries were tagmented and amplified using the Nextera XT kit

treatment_protocol: after overnight, diluted into fresh media, grown for 1 hour (WT_1hr_20220527)


title: HeLa cells, ITS O-MAP ChIP, Input, Rep2


title: Jurkat-NG-Lib1-Rep1

value: Jurkat-NG


title: HeLa cells, ITS O-MAP ChIP, Input, Rep1


title: SB791

description:
PETRI-seq; wildtype prolonged stationary phase (6 days) or standard overnight diluted into fresh media for 1 hour
Barcoded cDNA

treatment_protocol: after 6-day stationary growth, diluted into fresh media, grown 1 hour (d6_1hr_20220422); or after overnight, diluted into fresh media, grown for 1 hour (WT_1hr_20220527)


title: K562-LZ-Lib1-Rep1


title: SB780

description:
PETRI-seq; wildtype prolonged stationary phase (6 days) diluted into fresh media for 10 minutes - 1 hour or treated with ampicillin
Barcoded cDNA

treatment_protocol: after 6-day stationary growth, diluted into fresh media, grown for 10 minutes (d6_10min_1_20220422), 1 hour (d6_1hr_20220422, d6_1hr_2_20220422); or diluted into fresh media + ampicillin for 4 hours


title: SB768d

description:
PETRI-seq; wildtype stationary phase and 3 minutes - 1 hour after dilution
Barcoded cDNA

treatment_protocol: after overnight growth (WT_stat_20220331), diluted into fresh media, grown for 3 min (WT_3min_20220331), 10 min (WT_10min_20220331), 30 min (WT_30min_20220331), 1 hour (WT_1hr_20220331)


title: AKOC-2

city: hefei

first: Guangzhen

address: jinzhai road 96

department: Chemistry

institute: University of Science and Technology of China

data_processing:
Illumina Casava1.7 software used for basecalling.
Raw reads of fastq format were firstly processed using fastp and the low quality reads were removed to obtain the clean reads.
The clean reads were mapped to the reference genome using HISAT2. FPKM of each gene was calculated and the read counts of each gene were obtained by HTSeq-count
Assembly: mRatBN7.2
Supplementary files format and content: Matrix table with raw gene counts for every gene and every sample
Supplementary files format and content: Matrix table with FPKM values for every gene and every sample

organism: Rattus norvegicus

source_name: Liver

extract_protocol:
Total RNA was extracted using the TRIzol reagent (Invitrogen, CA, USA) according to the manufacturer’s protocol. RNA purity and quantification were evaluated using the NanoDrop 2000 spectrophotometer (Thermo Scientific, USA). RNA integrity was assessed using the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA).
The libraries were constructed using VAHTS Universal V6 RNA-seq Library Prep Kit according to the manufacturer’s instructions.

tag: tissue

value: Liver

value: AKOC


title: K562-Hifi-Lib1-Rep1


title: PHH_1343

city: Nijmegen

first: Haoyu

last: Wu

address: Mr. van Houtenstraat, 24

country: Netherlands

institute: Radboud University

data_processing:
Barcode information was extracted from read 1 and added to the ID of paired read 2 using UMI-tools 1.1.0. The barcoded reads containing unique molecular identifier (UMIs, 1-8bp) and cell barcode (9-16bp) were not used for mapping. Only the paired read 2 were aligned to a modified human genome (hg38) containing 92 ERCC spike-in sequences using STAR-2.7.6a with default settings. After mapping, featureCount 2.0.1 was used to assign the reads to genes. The number of UMIs per gene was counted for each cell and the gene-cell count tables were generated using UMI-tools 1.1.0 with the following parameters, --per-gene, --per-cell. For the downstream analysis, Seurat 3.2.3 was applied for data filtering, batch correction, normalization, clustering, visualization and differentially expressed gene analysis. Briefly, Seurat objects were created per donor using gene-cell matrices. Genes detected (UMIs > 0) in at least 5 cells were kept. Cells that are with less than 1000 UMIs or more than 50% of mitochondrial gene expression (mt%) were removed. UMI counts were then log2 transformed and normalized to sequencing depth per cell, and number of total UMIs and mt% were regressed out during the scaling to correct for sequencing depth and mt%.
For scRNA-seq data analysis within PHHs or ICOs, to minimize the batch effect, Seurat objects from different donors were integrated using the functions FindIntegrationAnchors and IntegrateData. Next, Top 2000 variable genes were found and used for PCA analysis with the setting, npcs=50. Seurat function FindNeighbors, FindClusters, and RunUMAP were used to construct a Shared Nearest Neighbor (SNN) Graph, identify clusters of cells based on the SNN graph, and finally to reduce the dimension for the visualization. Finally, differentially expressed genes from each cluster were identified, and the significant ones were used for pathway analysis via Enrichr web tool.
For integration analysis of PHHs and ICOs, PHH and ICO Seurat objects were merged using Seurat merge function. To compare ICOs to PHHs only, non-hepatocyte cluster in the PHHs dataset was not included in the integration. Then the newly merged Seurat object was first scaled with regressing out the number of total UMIs, mt% and biological replicates, and then used for further analysis following the same procedure as mentioned before.
For correlation analysis of zonally-expressed genes between human and mouse hepatocyte, we used a set of 94 genes previously identified to be expressed in both human and mouse. Average expression levels of each gene were calculated for all the 4 hepatocyte clusters (from this study) and then scaled and centered by z-scores. Same analysis was applied for nine layers of mouse hepatocytes. Pearson correlation was calculated to compare the 4 human hepatocyte clusters with the nine layers of mouse hepatocytes.
SCENIC R version was used for gene regulatory network analysis. Briefly, raw gene-cell count matrix was extracted from “RNA” assay in Seurat object and used as input for SCENIC. The matrix was log 2 transformed, and co-expression network was calculated using runGenie3 with default setting. To predict the transcription factor regulons, hg19-tss-centered-10kb-7species.mc9nr.feather motif database from RcisTarget was used. Regulons were created with the following parameters, minGenes = 20, coexMethod="top5perTarget". The Area Under the recovery Curve (AUC) scores were generated per cells and imported into Seurat for downstream analysis and visualization.
Genome_build: hg38

source_name: PHH

growth_protocol: Liver organoids were obtained from Erasmus university medical center and Radboud university medical center. Liver organoids were cultured and differentiated towards hepatocyte-like cells as published before (8, 18). Briefly, liver organoids were cultured in Basement Membrane Extract (BME, Trevigen, 3533-010-02) and the expansion medium was refreshed every 3 days. For the hepatocyte differentiation, the organoids were first cultured in expansion medium supplemented with BMP7 (PEPROTECH, 120-03P) for 5 days, and then switched to differentiation medium for more than 10 days. To obtain single cells, BME was removed by 10-fold dilution in cold DMEM/F-12 (Thermo, 12634-010). The liver organoids were then digested using 0.05% Trypsin-EDTA (Thermo, 25300-054) until 80-90% of the cells were single cells. Trypsin was removed and cells were collected for further use.

extract_protocol:
For single cell sorting, cells were washed one time with cold PBS containing 1% BSA, and then sorted using BDFACSMelody with a 100 µm nozzle into a 384-well plate containing 100 nl unique Cel-seq primer solution and 5 µl of mineral oil (Sigma, M5310).
Single-cell RNA libraries were constructed according to the mCEL-seq2 protocol . Briefly, 100 nL lysis buffer (0.2% Triton X-100, 2mM dNTPs, 1:50.000 ERCC ExFold RNA spike-in (Thermo, 4456739)) was added per well using the Nanodrop Ns-2 Stage (BioNex). For the in vitro reverse transcription reaction, SuperScript™ II Reverse Transcriptase (Invitrogen, 18064-14) was used with the following the protocol, 4 ⁰C for 5 min; 25 ⁰C for 10 min; 42 ⁰C for 60 min; 70 ⁰C for 10 min. Next, 960 nL second-strand reaction mix containing E. coli DNA ligase (NEB, M0205L), E. coli DNA polymerase I (NEB, M0209L) and Random hexamer (sigma, 11034731001) were added per well for second-strand synthesis following the protocol, 16 ⁰C for 2 hrs. All the cDNA from one 384-well plate was pooled. In vitro transcription was performed using the MEGAscript™ T7 Transcription Kit (Invitrogen, AMB 1334-5) by incubating the reaction at 37 ⁰C for 14 hrs. The amplified RNA was fragmented and used for cDNA synthesis using Superscript II and the random octamer primer. Libraries were amplified for 9 cycles for the hepatocyte libraries and 8 cycles for the liver organoid libraries using Phusion® High-Fidelity PCR Master Mix (NEB, M0531S) with the Nextflex primers, and quantified on the bio-analyzer using DNA high-sensitivity kit (Agilent, 5067-4626). Barcoded libraries were sequenced on an Illumina NextSeq 500 with 38bp paired-end reads at a depth of about 25 million reads per plates.

treatment_protocol: no treatment

value: PHH

tag: plate index


title: SB767d

tag: cell type


title: Jurkat-Hifi-Lib1-Rep1

value: T lymphocyte cells

tag: treatment

value: Jurkat-Hifi


title: SB766d


title: AKOC-1


title: Quik-clot-3

value: Quik-clot


title: K562-Sniper-Lib1-Rep2

value: myelogenousleukemiacell


title: Jurkat-LZ-Lib1-Rep1

value: Jurkat-LZ


title: Quik-clot-2


title: Jurkat-Sniper-Lib1-Rep2

value: Jurkat-Sniper


title: PHH_1339


title: PHH_1340


title: Quik-clot-1


title: SB787

value: wildtype


title: HEK293T_Control_2

value: control


title: SB760d

description:
PETRI-seq; wildtype cells in lag phase
Barcoded cDNA

treatment_protocol: after overnight growth, diluted into fresh media, grown for 10 minutes (WT_10min_20220210)


title: PHH_1342


title: PHH_1338


title: SB765d


title: AKOC-3


title: HEK293T_Control_1


title: SB746d


title: Mid_rep2

city: Boston

first: Kornelia

last: Polyak

state: MA

address: 450 Brookline Ave

department: Medical Oncology

institute: Dana-Farber Cancer Institute

description: 10x Genomics

data_processing:
Demultiplexing, barcoded processing, feature counting and aggregation was done using Cell Ranger 6.1.2.
Assembly: Rnor_6
Supplementary files format and content: Supplementary files format and content: tsv files and matrix files

source_name: mammary gland

extract_protocol:
Mammary glands were collected freshly and digested in digestion medium containing 2 mg/mL collagenase type IV, 2 mg/mL hyaluronidase and 2 mg/mL BSA in DMEM/F12 medium, at 37 ºC for 1 to 2 hours, then viable single cells were submitted to Translational Immunogenomics Lab core in Dana-Farber cancer institute for library construction.
The scRNA-seq libraries were prepared according to the Chromium Next GEM Single Cell 5’ HT v2 protocol (10x Genomics). Quality control for amplified cDNA libraries and final sequencing libraries were performed using Bioanalyzer High Sensitivity DNA Kit (Agilent). The pooled libraries were sequenced on Illumina NovaSeq S4 platform.

treatment_protocol: For midkine treatment, rat recombinant midkine was injected intraperitoneally 3 times/week at 300 ug/kg for four consecutive weeks. All rats were sacrificed immediately after the final dose, and mammary tissues were collected for scRNA sequencing.

value: mammary gland

value: rat recombinant midkine protein


title: K562-LZ-Lib1-Rep2


title: PHH_1337


title: K562-NG-Lib1-Rep2


title: PHH_1336


title: SB745


title: Jurkat-NG-Lib1-Rep2


title: PHH_1341


title: PHH_1335


title: SB722d

description:
PETRI-seq; hipA7 cells after dilution from overnight
Barcoded cDNA

treatment_protocol: after overnight growth, diluted into fresh media, grown for 50 minutes


title: Veh_rep2

description: 10x Genomics

value: vehicle


title: Jurkat-LZ-Lib1-Rep2


title: Mid_rep1

description: 10x Genomics


title: K562-Hifi-Lib1-Rep2


title: Veh_rep1

description: 10x Genomics


title: ICO3-EM_974

source_name: ICO

value: ICO


title: ICO3-EM_975


title: SB690

description:
PETRI-seq; hipA7, metG*, and wildtype cells after dilution from overnight
Barcoded cDNA

extract_protocol:
in situ RT and ligations followed by cell lysis with detergent and proteinase K
Double-stranded cDNA was made by second strand synthesis. Libraries were tagmented and amplified using the Nextera XT kit. rRNA was depleted using GenScript Cas9 and partial probe set

treatment_protocol: after overnight growth, diluted into fresh media, grown for 10 minutes-52 minutes

value: hipA7, metG, wildtype


title: ICO3-DM_973


title: SRSLY_BR_V_S

city: Cleveland

first: Hyunmin

last: Kim

state: Ohio

address: 0900 Euclid Ave.

institute: Case Western Reserve University

data_processing:
trim fastq using fastp with Qxyg option
map the trimmed reads to galGal6 genome
extract mapped alignments in bed format using bedtools
Assembly: galGal6
Supplementary files format and content: bed coordinates and mapping read sequences

organism: Brachylophosaurus canadensis

source_name: vessels

extract_protocol:
Tyrannosaurus rex (MOR1125) and Brachylophosaurus canadensis (MOR 2598) were prepared and analyzed in separate laboratories from the extant material, with separate buffers, reagents and instruments, by investigators wearing all protective lab garb (coats, hair covers, masks, gloves, etc.) and following aseptic procedures. Samples were prepared in the BioGard hood. Cortical bone fragments from each dinosaur were subjected to demineralization by incubation in 0.5 M EDTA (pH 8.0). Debris remaining from demineralization was filtered through a 40 µm cell strainer (BD Falcon 352340), and filtrate was centrifuged and washed with E-pure water to collect cells. Vessels were isolated from debris trapped within the cell strainer and washed. Both cells and vessels were incubated with the iron chelator pyridoxal isonicotinoyl hydrazone (PIH) in solution (5 mM PIH in 50 mM NaOH) (1,2,3) to remove iron, and then were prepared with "Protocol for preparing non-mammalian samples for Omni-C sequencing and do novo genome reconstruction— 2/22/2021, adopted by Berkley Gryder from Dovetail protocol used in the DNA zoo project" beginning at step 3, as above.
KAPA HyperPlus Kit protocol (Roche 07962401001)

value: vessels


title: Jurkat-Hifi-Lib1-Rep2


title: M22_rep2

description: 10x Genomics

value: 22 months


title: SB691d


title: SB665d

description:
PETRI-seq; wildtype cells in tetracycline
Barcoded cDNA

treatment_protocol: after overnight growth, diluted into fresh media + tetracycline (tet1_20211014, tet5_20211014); or after overnight growth, diluted into fresh media, grown for 50 minutes (tet2_20211014), tetracycline added for 30 minutes (tet3_20211014)


title: M22_rep3

description: 10x Genomics


title: SB692d


title: SRSLY_BR_O_S

source_name: osteocytes

value: osteocytes


title: SB646

description:
PETRI-seq; wildtype bioreactor timepoints (undiluted/stationary, 20 minutes - 2 hours after dilution)
Barcoded cDNA

treatment_protocol: after chemostat exponential growth; cells taken 2 (t1), 2.9 (t2), 4 (t3), or 5.5 (t4) hours after OD600=0.1; sequenced in stationary phase or 20 minutes-2 hours after dilution into fresh media


title: ICO3-DM_972


title: SRSLY_BR_V


title: ICO2-EM_1067


title: M12_rep4

description: 10x Genomics

value: 12 months


title: ICO2-DM_1065


title: SB664d


title: M22_rep1

description: 10x Genomics


title: KAPA_BR_V


title: SB642d2

extract_protocol:
in situ RT and ligations followed by cell lysis with detergent and proteinase K
Double-stranded cDNA was made by second strand synthesis. Libraries were tagmented and amplified using the Nextera XT kit. rRNA was depleted using GenScript Cas9 and partial probe set two times


title: ICO1-EMDM_1344


title: SRSLY_BR_O


title: M22_rep4

description: 10x Genomics


title: SB640


title: SB641d


title: M12_rep2

description: 10x Genomics


title: ICO1-EM_1543


title: M6_rep3

description: 10x Genomics

value: 6 months