title: Rotenone_SHSY5Y_rep3_rotData

first: Thomas

middle: F.

last: Meyer

address: Schwanenweg 20, Haus U27, 2 OG

department: Infektion Onkologie

institute: IKMB

description:
Library name: Rotenone_SHSY5Y_rep3_rotData
Rotenone 10uM of Rotenone dataset

data_processing:
Quality of raw reads was analyzed by fastqc. In order to obtain the matrix of gene counts, a custom pipeline written using the python workflow manager Snakemake available on https://github.com/aqvd/snakemake_workflows/blob/main/Chip-Seq/Snakefile_RNAseq.py was used. Briefly, raw reads are mapped to the Human reference genome (GRCh38) using hisat2. Alignments with both pairs aligned in the proper orientation are used to quantify gene expression using htseq-count. Gene annotation from Ensembl (release 97) was used. The matrix with gene expression counts created from htseq-count was used as starting point for Differential Expression Analysis using the DESeq2 R package (v 1.40.2).
Assembly: GRCh38.p12
Supplementary files format and content: *.bam files contain the coordenate sorted hisat2 alignments against GRCh38.p12 genome assembly
Supplementary files format and content: raw_counts.csv is the Gene Count matrix output of htseq count using Ensembl v102 genome annotation

source_name: Bone; Marrow

growth_protocol: SH-SY5Y cells (ATCC, CRL-2266, RRID: CVCL_0019) were maintained at 37°C in a 5% CO2 humidified incubator. SH-SY5Y cells were cultured in medium composed of 40% MEM (#31095029, Thermo Fisher), 40% Ham's F-12 Nutrient Mix (#31765068, Thermo Fisher),15% FBS (#10,437,028, Thermo Fisher), 1× MEM Non-Essential Amino Acids Solution (#11140050, Thermo Fisher), 25mM HEPES, 1mM sodium pyruvate (#11360039, Thermo Fisher). To split the cells, accutase (#00-4555-56, Thermo Fisher) was used to detach the cells. For Hela cells, DMEM (#DMEM-HPSTA, Capricon), 10% heat-inactivated FBS and 25 mM HEPES (#HEP-B, Capricon) was used to prepare the growth medium.

extract_protocol:
RNeasy Plus Mini Kit (Qiagen#74136)
the sequencing of the samples was performed using the PE150 sequencing strategy on NovaSeq X Plus (Illumina, San Diego) by Novogene UK (Cambridge, UK). Messenger RNA (mRNA) 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: Prior to each experiment, cells were starved in medium containing no FBS for 2 hours.  For the Helicobacter pylori (Hpy) Dataset: Helicobacter pylori P12 WT strain is enriched in bacto brain hear fusion (#237500, BD) with 10% heat-inactivated FBS is grown on hpGC agar (#CM0367, Oxoid) plate containing 10% heat-inactivated horse serum and 10% vitamin mix. Bacteria were re-streaked after 2-3 days and they were used for infection 16-24 hours after re-streaking. The streaked plates were incubated at 37°C under microaerophilic conditions for 24 h.SH-SY5Y cells were infected with Hpy at a MOI of 100 (Hpy), or pre-treated with 200 nM ponatinib and then infected with H. pylori (Hpy - Ponatinib). Vehicle only (DMSO) and Ponatinib without infection (Ponatinib) controls were performed.  For the Rotenone Dataset:  Rotenone (10 µM, #R8875, Sigma-Aldrich) was used to treat SH-SY5Y cells. For the co- treatment cells were treated with ponatinib 50 nM (Seleckchem, #S1490) one hour before rotenone (10 µM) treatment. Vehicle only (DMSO) and Ponatinib only controls were included.

value: Bone; Marrow

value: neuroblastoma

value: Rotenone


title: Carotid Artery, CDH5cre negative Cx43fl/fl, 18hr post-injury

status: Public on Sep 24 2025

type: SRA

city: Roanoke

first: Mark

middle: Colin

last: Renton

state: VA

address: 4 Riverside Circle, Room 2208

department: Fralin Biomedical Research Institute

country: USA

institute: Virginia Tech

description:
Library name: CDH5neg
Novogene 10X single cell 3' RNA Analysis

library_source: transcriptomic single cell

data_processing:
Raw sequencing data in FASTQ format were pre-processed with Cell Ranger (v8.0.1) using default parameters, and subsequent reads were aligned to the mouse genome using the STAR aligner within Cell Ranger.
Assembly: Mouse mm10 (Cell Ranger mm10-2020-A)
Supplementary files format and content: Tab-separated values files and matrix files

molecule: total RNA

organism: Mus musculus

source_name: Carotid Artery

extract_protocol:
Both EC-Cx43 KO (CDH5cre positive) and EC-WT (CDH5cre negative)  mice were euthanized by CO2 overdose and cervical dislocation 18 hr post carotid EC injury and the injured carotid was exposed without perfusion to minimize time and disruption to the endothelial layer. The carotid was then removed from the mouse and dipped in a solution of heparin saline followed by a saline-only solution. Before digestion, carotids were stored in ice-cold EC media (Vascular Cell Basal Medium; ATCC #PCS-100-030) supplemented with all components of an Endothelial Cell Growth Kit (ATCC #PCS-100-041) excluding fetal bovine serum (FBS) while subsequent carotids were removed. To enrich for EC while collecting enough total cells for processing and sequencing, carotids were cut open lengthwise prior to removal to expose the EC layer for digestion. Carotids were kept whole and a short 20 min digest (described below) was performed.  Up to two carotids were digested in a 1 mL digestion solution of 0.2 mg/ml liberase TM (Roche #5401119001) and 60 U/ml DNase1 in supplemented EC media. Carotids were incubated at 37°C and 400 rpm in a benchtop orbital shaking heat block for 20 min, pipetting up and down with a 1 mL pipette every 5 min. Digestion was stopped with the addition of 1 mL stopping solution (2% FBS, 1mM EDTA, in supplemented EC media), after which samples of the same condition were combined (up to 5 at a time to reduce time to digestion). A 100 μm cell strainer was primed by flushing through 2 mL of supplemented EC media with 2% FBS. Cell solutions were filtered through the strainer and rinsed with 3 mL of supplemented EC media with 2% FBS. The flow through was centrifuged at 1000 rpm, 4°C for 10 min, followed by removal of the supernatant and resuspension of the cell pellet in 2 mL RBC lysis buffer (Invitrogen 00-4333-57) at room temperature for 5 min. RBC lysis was stopped using 10 ml supplemented EC media with 2% FBS, samples were centrifuged at 1000 rpm, 4°C for 10 min and the cell pellet was resuspended in 10% DMSO, 10% FBS in cold supplemented EC media. Single cell suspensions were slowly frozen at -80°C overnight in a cell freezer container, then transferred to liquid nitrogen until sequencing. A total of 15 carotids for EC-WT and 20 carotids for EC-Cx43 KO were pooled for use for scRNAseq by Novogene.
Novogene scRNAseq Methods. Cell viability analysis, RNA integrity analysis, library preparation, sequencing, raw read count generation, and reference genome alignment were performed commercially by Novogene (Sacremento, CA, USA) using their Plant and Animal 10X Single Cell Transcriptome Sequencing pipeline. Full parameters, protocols, and quality control reports are available on request, but are explained briefly below:  Sample and Library Preparation. Cryopreserved cell samples were thawed and processed by Novogene. Cell count and viability were assessed using a TC20 Automated Cell Counter (Bio-Rad, USA) with both samples achieving approximately 60,000 cells with an overall cell viability of >75%. Libraries were then constructed using the 10x Chromium Single Cell 3’ Kit with a target of 10,000 cells.  Sequencing and Data Preprocessing. Sequencing was performed on the NovaSeq X Plus Series (Illumina, USA) platform with 150 bp paired end reads with a minimum of 150 G raw data per sample.

tag: tissue

value: Carotid Artery

tag: genotype

value: CDH5 Cre- Cx43fl/fl

tag: treatment

value: 18 hr ligation injury recovery


title: Rotenone_SHSY5Y_rep2_rotData

city: Kiel

country: Germany

description:
Library name: Rotenone_SHSY5Y_rep2_rotData
Rotenone 10uM of Rotenone dataset

library_source: transcriptomic

molecule: polyA RNA

organism: Homo sapiens

tag: cell line

tag: cell type

tag: batch


title: Rotenone_SHSY5Y_rep1_rotData

description:
Library name: Rotenone_SHSY5Y_rep1_rotData
Rotenone 10uM of Rotenone dataset


title: Cell-line 1, Sppl3/Mgat2/St3gal3/4/6/B3gnt2 KO, cell-line1, rep1

city: La Jolla

first: Nathan

middle: E.

last: Lewis

state: CA

address: 9500 Gilman Dr. IBRFII, 5220E

department: Bioengineering; Pediatrics

institute: University of California, San Diego

description:
Library name: CL1_R1
B12_R1

data_processing:
The samples were quality controlled using FastQC and the adapter sequences trimmed using Trimmomatic
The sequences were demultiplexd with bcl2fastq v2.20 Conversion Software.
Reads were mapped to the CHO GCF_003668045.1_CriGri-PICRR genome, aligned and quantified with Kallisto.
Assembly: CHO GCF_003668045.1_CriGri-PICRR
Supplementary files format and content: csv files includes raw counts for sample
Supplementary files format and content: cvs files include TPM values for each sample

organism: Cricetulus griseus

source_name: CHO-S

growth_protocol: The geCHO cells were grown in 125 ml Erlenmeyer shaker flasks with baffled bottom (Fisher Scientific, USA) at 130 rpm in 37°C at 5% CO2 with maintenance media containing CD CHO Medium (Gibco, USA) supplemented with 8 mM L-Glutamine (Gibco) and 1x anti-clumping agent (Gibco). The geCHO cells expressing E1E2 and Furin were grown in media containing 1 mg/ml Geneticin Selective Antibiotic (G418 sulfate; Gibco) and 0.75 mg/ml Hygromycin B (Gibco) for selection.

extract_protocol:
RNA was harvested and extracted using the RNAeasy Kit (Qiagen)
RNA quality was analyzed using the Agilent Tapestation 4200, and only samples with an RNA Integrity Number (RIN) exceeding 8.0 were selected for library construction. Libraries were prepared using the Illumina Stranded mRNA Sample Prep Kit with Illumina RNA UD Indexes (Illumina, San Diego, CA), following the manufacturer's guidelines.

treatment_protocol: The geCHO cells were initially transfected with the furin plasmid and cloned. The cloned furin cell lines were then transfected with sE1E2.SZ-H445P plasmid and cloned again.

value: CHO-S

value: Chinese Hamster Ovaries

value: Sppl3/Mgat2/St3gal3/4/6/B3gnt2 KO


title: DM-DDX-4_S1206

city: MADISON

first: Siqi

last: Shen

state: WI

address: 425 Henry Mall

institute: University of Wisconsin - Madison

description: Library name: DM-DDX-4_S1206

data_processing:
Data are aligned by STAR-2.7.3a. RSEM is used to generate results files.
Assembly: mm39
Supplementary files format and content: RSEM reusults file

growth_protocol: Cells were cultured in OPTI-MEM supplemented with 10% FBS, 1% penicillin-streptomycin, 1% SCF-conditioned medium, 28.6 μM β-mercaptoethanol, 1 μM b-estradiol, and 400 μg/ml G418 (immortalized cell culture media). Cells were cultured in a humidified 5% CO2 incubator at 37˚C.

extract_protocol:
Total RNA was purified from 2-5 x 10^5 cells with TRIzol (Invitrogen) and 1-2 mg RNA was treated with DNase I (Thermo Fisher Scientific) for 15 min at room temperature. After heat inactivation of DNase I with EDTA for 10 min at 65°C, 0.5-1 mg RNA was incubated with a 4:1 mixture of oligo(dT) primers and random hexamer at 68°C for 10 min. RNA/primers were incubated with Moloney murine leukemia virus reverse transcription (M-MLV RT) (Thermo Fisher Scientific), 5X first strand buffer (Thermo Fisher Scientific), 10 mM dithiothreitol (Thermo Fisher Scientific), RNAsin (Promega), and 0.5 mM deoxynucleoside triphosphates (New England Biolabs) at 42°C for 1 h and then heat inactivated at 95°C for 5 min.
RNA library was prepared using TruSeq Stranded Total RNA (rRNA depletion) Library kit following manufacturer's protocol.

value: HoxB8-immortalized myeloid progenitor cell

value: infected with GFP-DDX41 expressing retrovirus


title: C3KO-E-4_S1207

description: Library name: C3KO-E-4_S1207

value: infected with GFP-expressing retrovirus


title: Het-DDX-4_S1204

description: Library name: Het-DDX-4_S1204

source_name: Het-DDX

value: Het-DDX


title: Rotenone-Ponatinib_SHSY5Y_rep1_rotData

description:
Library name: Rotenone-Ponatinib_SHSY5Y_rep1_rotData
Rotenone + ponatinib of Rotenone dataset

value: Rotenone-Ponatinib


title: DM-E-4_S1205

description: Library name: DM-E-4_S1205


title: C3KO-E-3_S1201

description: Library name: C3KO-E-3_S1201


title: Ponatinib_SHSY5Y_rep3_rotData

description:
Library name: Ponatinib_SHSY5Y_rep3_rotData
Ponatinib only of Rot dataset

value: Ponatinib


title: DM-DDX-3_S1200

description: Library name: DM-DDX-3_S1200


title: Ponatinib_SHSY5Y_rep2_rotData

description:
Library name: Ponatinib_SHSY5Y_rep2_rotData
Ponatinib only of Rot dataset


title: DM-E-3_S1199

description: Library name: DM-E-3_S1199


title: Ponatinib_SHSY5Y_rep1_rotData

description:
Library name: Ponatinib_SHSY5Y_rep1_rotData
Ponatinib only of Rot dataset


title: Het-DDX-3_S1198

description: Library name: Het-DDX-3_S1198


title: DMSO_SHSY5Y_rep3_rotData

description:
Library name: DMSO_SHSY5Y_rep3_rotData
DMSO control of Rot dataset

value: DMSO


title: DMSO_SHSY5Y_rep2_rotData

description:
Library name: DMSO_SHSY5Y_rep2_rotData
DMSO control of Rot dataset


title: C3KO-E-2_S1195

description: Library name: C3KO-E-2_S1195


title: Het-E-3_S1197

description: Library name: Het-E-3_S1197

source_name: Het-E

value: Het-E


title: Cell-line 1, Sppl3/Mgat2/St3gal3/4/6/B3gnt2 KO, E1E2, Furin,cell-line1_17, rep3

description:
Library name: CL1_17_R3
B12_17_R3


title: DMSO_SHSY5Y_rep1_rotData

description:
Library name: DMSO_SHSY5Y_rep1_rotData
DMSO control of Rot dataset

value: Bone; Marrow


title: DM-DDX-2_S1194

description: Library name: DM-DDX-2_S1194


title: Rotenone-Ponatinib_SHSY5Y_rep3_rotData

description:
Library name: Rotenone-Ponatinib_SHSY5Y_rep3_rotData
Rotenone + ponatinib of Rotenone dataset

value: neuroblastoma


title: Ponatinib_SHSY5Y_rep3_hpyData

description:
Library name: Ponatinib_SHSY5Y_rep3_hpyData
Ponatinib only of Hpy dataset


title: DM-E-2_S1193

description: Library name: DM-E-2_S1193


title: Het-E-4_S1203

description: Library name: Het-E-4_S1203


title: Rotenone-Ponatinib_SHSY5Y_rep2_rotData

description:
Library name: Rotenone-Ponatinib_SHSY5Y_rep2_rotData
Rotenone + ponatinib of Rotenone dataset


title: Ponatinib_SHSY5Y_rep1_hpyData

description:
Library name: Ponatinib_SHSY5Y_rep1_hpyData
Ponatinib only of Hpy dataset


title: Ponatinib_SHSY5Y_rep2_hpyData

description:
Library name: Ponatinib_SHSY5Y_rep2_hpyData
Ponatinib only of Hpy dataset


title: Het-E-2_S1191

description: Library name: Het-E-2_S1191


title: Hpy_SHSY5Y_rep2_hpyData

description:
Library name: Hpy_SHSY5Y_rep2_hpyData
Hpy infection of Hpy dataset

value: Hpy


title: DM-DDX-1_S1188

description: Library name: DM-DDX-1_S1188


title: C3KO-E-1_S1189

description: Library name: C3KO-E-1_S1189


title: Hpy_SHSY5Y_rep1_hpyData

description:
Library name: Hpy_SHSY5Y_rep1_hpyData
Hpy infection of Hpy dataset


title: Het-DDX-1_S1186

description: Library name: Het-DDX-1_S1186


title: Hpy-Ponatinib_SHSY5Y_rep2_hpyData

description:
Library name: Hpy-Ponatinib_SHSY5Y_rep2_hpyData
Hpy + Ponatinib of Hpy dataset

value: Hpy-Ponatinib


title: DM-E-1_S1187

description: Library name: DM-E-1_S1187


title: Hpy-Ponatinib_SHSY5Y_rep1_hpyData

description:
Library name: Hpy-Ponatinib_SHSY5Y_rep1_hpyData
Hpy + Ponatinib of Hpy dataset


title: MC38_Vector

city: Beijing

first: Shengde

last: Liu

address: 38 Xueyuan Rd , Haidian District

country: China

institute: Peking University Health Science Center

description:
Library name: Sample 1
Single-cell RNA sequencing

data_processing:
The demultiplexing, barcoded processing, gene counting and aggregation weremade using the Cell Ranger software v2.1.1(https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/what-is-cell-ranger
Assembly: Mus_musculus.GRCm39.110
Supplementary files format and content: Tab-separated values files and matrix files

source_name: tumor

extract_protocol:
For single-cell RNA sequencing (scRNA-seq) of subcutaneous tumors, a stable MC38 cell pool expressing SPP1 or a control vector was established via lentiviral transduction. Western blotting was performed to confirm successful expression of SPP1. These cells were then subcutaneously injected into C57BL/6 mice for tumor formation. Seven days later, mice in the OE-SPP1 and OE-vector groups were intraperitoneally injected with anti-PD1 (10 mg/kg, twice a week). After two weeks of treatment, the mice were euthanized, and tumor tissues were carefully excised. The tumor tissues were dissociated into single-cell suspensions using enzymatic digestion (e.g., collagenase and DNase), followed by mechanical dissociation. The resulting cell suspension was filtered through a 70 µm cell strainer to remove large debris and dead cells.
Single-cell libraries were prepared using nova X plus, which allows for high-throughput single-cell capture and barcoding. The captured cells were encapsulated into droplets, where reverse transcription and cDNA amplification occurred. After library construction, sequencing was performed on a high-throughput platform (nova X plus), with a minimum depth of 20,000 reads per cell.

value: tumor

value: colon adenocarcinoma cells

value: WT

value: anti-PD-1 (10 mg/kg, twice a week)


title: Het-E-1_S1185

description: Library name: Het-E-1_S1185


title: MC38_SPP1

description:
Library name: Sample 2
Single-cell RNA sequencing


title: DMSO_SHSY5Y_rep2_hpyData

description:
Library name: DMSO_SHSY5Y_rep2_hpyData
DMSO control of Hpy dataset


title: Hpy-Ponatinib_SHSY5Y_rep3_hpyData

description:
Library name: Hpy-Ponatinib_SHSY5Y_rep3_hpyData
Hpy + Ponatinib of Hpy dataset


title: Hpy_SHSY5Y_rep3_hpyData

description:
Library name: Hpy_SHSY5Y_rep3_hpyData
Hpy infection of Hpy dataset


title: DMSO_SHSY5Y_rep3_hpyData

description:
Library name: DMSO_SHSY5Y_rep3_hpyData
DMSO control of Hpy dataset


title: CITEseq, DC_R2, FBC

city: Bethesda

first: Can

last: Li

state: Maryland

address: 9000 Rockville Pike

institute: NIH

description: Library name: DC_R2_adt

library_source: other

data_processing:
Base calling was performed using RTA 3.4.4
Demultiplexing was performed using cellranger v8.0.1 (Bcl2fastq 2.20.0)
Alignment was performed using cellranger v8.0.1 (STAR 2.7.2a)
Assembly: refdata-gex-GRCm39-2024-A
Supplementary files format and content: SCAF4218_DC_R1_filtered_feature_bc_matrix.h5
Supplementary files format and content: SCAF4219_DC_R2_filtered_feature_bc_matrix.h5
Library strategy: CITE-seq

molecule: protein

source_name: Lymph node

extract_protocol:
DCs sorted from pLNs of C57BL/6 mice. The cellular samples were loaded in the lanes according to the 10X Genomics Chromium Next GEM Single Cell 3’v4 (CG000732 Rev B) User Guide with a single capture lane per sample.
3’ v4 gene expression profiling and feature barcode libraries (Total Seq-B)  were performed as described in the vendor provided user guide.

value: Lymph node

value: Dendritic cells

tag: strain


title: DMSO_SHSY5Y_rep1_hpyData

description:
Library name: DMSO_SHSY5Y_rep1_hpyData
DMSO control of Hpy dataset


title: NCI-H358-R cell line resistant to sotorasib, under sotorasib pressure, rep3

city: Boston

first: Alexander

last: Ivliev

state: MA

address: 645 Summer Street, Suite 101

institute: Ikena Oncology, Inc.

description: H358-Resistant-250nM-Sotorasib-4days-Rep3

data_processing:
Image analysis and base calling were conducted by the Control software. Raw sequence data (.bcl files) generated the sequencer were converted into fastq files and de-multiplexed using Illumina's bcl2fastq 2.17 software. One mismatch was allowed for index sequence identification.
Quality of fastq files was assessed using fastqc software (v0.12.1).
Counts and transcript per million (TPM) were generated from fastq files using Salmon (v0.14.1) with the “validateMappings”, “gcBias”, and “seqBias” flags on. Salmon index was based on GENCODE human transcriptome (Release 38, GRCh38.p13) with the entire human genome sequence used as decoy.
Tximport R package (1.28.0) was used to collect counts and TPM values across samples into respective matrices.
Assembly: GENCODE human transcriptome (Release 38, GRCh38.p13)
Supplementary files format and content: tab-delimited text file includes raw counts for each Sample
Supplementary files format and content: tab-delimited text file includes transcripts per million (TPM) for each Sample

growth_protocol: NCI-H358 cell line was purchased from ATCC and grown in RPMI-1640 Medium supplemented with 10% fetal bovine serum (FBS). The H358-R cell line was created by continuous culturing of the H358 cells in sotorasib (250 nM) until cells grew readily under sotorasib pressure (approximately 2 months). NCI-H358-R cells were grown in RPMI-1640 Medium supplemented with 10% FBS and 250 nM sotorasib.

extract_protocol:
RNA extraction, library preparation, sequencing, and analysis was conducted at Azenta Life Sciences (South Plainfield, NJ, USA) as follows. Total RNA was extracted from frozen cell pellet samples using Qiagen RNeasy Plus Universal micro kit following manufacturer’s instructions (Qiagen, Hilden, Germany).
RNA samples were quantified using Qubit 2.0 Fluorometer (Life Technologies, Carlsbad, CA, USA) and RNA integrity was checked using Agilent TapeStation 4200 (Agilent Technologies, Palo Alto, CA, USA).  RNA sequencing libraries were prepared using the NEBNext Ultra II RNA Library Prep Kit for Illumina using manufacturer’s instructions (NEB, Ipswich, MA, USA). Briefly, mRNAs were initially enriched with Oligod(T) beads. Enriched mRNAs were fragmented for 15 minutes at 94 °C. First strand and second strand cDNA were subsequently synthesized. cDNA fragments were end repaired and adenylated at 3’ends, and universal adapters were ligated to cDNA fragments, followed by index addition and library enrichment by PCR with limited cycles. The sequencing library was validated on the Agilent TapeStation (Agilent Technologies, Palo Alto, CA, USA), and quantified by using Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA) as well as by quantitative PCR (KAPA Biosystems, Wilmington, MA, USA).  The sequencing libraries were clustered on a flowcell. After clustering, the flowcell was loaded on the Illumina NovaSeq instrument according to manufacturer’s instructions. The samples were sequenced using a 2x150bp Paired End (PE) configuration.

tag: resistance

value: resistant to sotorasib

value: sotorasib (250 nM, profiled 4 days after dosing)


title: CITEseq, DC_R2, GEX

description: Library name: DC_R2_mRNA


title: CITEseq, DC_R1, FBC

description: Library name: DC_R1_adt


title: NCI-H358-R cell line resistant to sotorasib, under sotorasib pressure, rep2

description: H358-Resistant-250nM-Sotorasib-4days-Rep2


title: CITEseq, DC_R1, GEX

description: Library name: DC_R1_mRNA


title: Het-DDX-2_S1192

description: Library name: Het-DDX-2_S1192

value: Het-DDX

value: HoxB8-immortalized myeloid progenitor cell

value: infected with GFP-DDX41 expressing retrovirus


title: NCI-H358-R cell line resistant to sotorasib, under sotorasib pressure, rep1

description: H358-Resistant-250nM-Sotorasib-4days-Rep1


title: NCI-H358-R cell line resistant to sotorasib, no sotorasib pressure, rep3

value: no treatment


title: NCI-H358-R cell line resistant to sotorasib, no sotorasib pressure, rep2


title: PBS

city: changchun

first: Man

last: Sun

address: No. 2699, Qianjin Street, Chaoyang District, Changchun City, Jilin Province, China

institute: jilin university

description: Library name: sample A

data_processing:
scRNA-Seq
Assembly: rattus_norvegicus__ensembl-97
Supplementary files format and content: Tab-separated values files and matrix files

source_name: Pancreatic cancer

extract_protocol:
Single-cell suspensions were prepared according to the protocol of Chromium Single Cell 3’Solution (V3 chemistry). All specimens were washed two times with cold 1x phosphate-buffered saline (PBS) .Haemocytometer (Thermo Fisher Scientific) was used to evaluate cell viability rates.Then, we used Countstar to count the concentration of sinale-cell suspension and adiust the concentration to1000 cells/ul. Samples that were lower than the required cell concentration defined in the user guide (i.e., <400 cells/ul) were pelleted and re-suspended in a reduced volume, and then the concentration of the new solution was counted again.
Single-cell RNA sequencing (scRNA-seq) libraries were constructed using the 10x Chromium Single Cell 3’ platform (10X Genomics, Pleasanton, CA, USA) according to the manufacturer’s protocol. Briefly, single-cell suspensions were loaded onto a Chro-mium microfluidic chip to generate single-cell Gel Beads in Emulsion (GEMs). The scRNA-seq libraries were then prepared using the Chromium Single Cell 3’ Reagent Kit v3.1 (10X Genomics, Pleasanton, CA, USA). Library quality was assessed using the Ag-ilent 2100 Bioanalyzer system (Agilent Technologies, Santa Clara, CA, USA), and se-quencing was performed on the Illumina NovaSeq 6000 platform (Illumina, San Di-ego, CA, USA) with a sequencing depth of at least 50,000 reads per cell.

value: Pancreatic cancer

value: PBS


title: NCI-H358-R cell line resistant to sotorasib, no sotorasib pressure, rep1


title: EpH4 Cldn-null_2

city: Kyoto

first: Kazuto

last: Tsukita

state: Kyoto

address: 54 Shogoin-Kawaharacho

country: Japan

institute: Kyoto university

data_processing:
or the processing of RNAseq data, adapter sequences were first removed, and reads were quality-trimmed using Trimmomatic (version 0.36). The trimming process involved removing Nextera adapter sequences, trimming low-quality bases from both the leading and trailing ends, and applying a sliding window trimming approach. The minimum length threshold for reads after trimming was set to 50 bases. Post-trimming, the quality of the reads was assessed using FastQC (version 0.11.9). The reference genome for Mus musculus (mm10) was downloaded from the Ensembl database and used for subsequent alignment and gene expression quantification. Trimmed reads were aligned to the reference genome using STAR (version 2.7.9a) with the aligner configured to run in paired-end mode utilizing 10 threads. The output files included sorted BAM files and transcriptome alignments for downstream analysis. Gene expression levels were quantified using RSEM (version 1.3.3), with quantification performed in paired-end mode, specifying reverse strandedness.
Assembly: mm10
Supplementary files format and content: comma-delimited file includes the raw counts for each sample.

growth_protocol: EpH4 epithelial cells were maintained in DMEM supplemented with 10% fetal bovine serum (FBS), 2mM L-glutamine, and 0.45% (w/v) D (+) glucose at 37˚C with 5% CO2

extract_protocol:
RNA was harvested using RNeasy Mini Kit (QIAGEN).
RNA libraries for RNA-seq were prepared using SMART-Seq v4 Ultra Low Input RNA Kit for sequencing (Takara Bio), Nextera XT DNA Library Prep Kit and IDT for Illumina - DNA/RNA UD Indexes, Tagmentation (Illumina) following manufacture's protocols.

value: mouse mammary gland epithelial cells

value: Cldn-null

tag: time

value: Day 7


title: EpH4 Cldn-null_1


title: EpH4 WT_3


title: EpH4 WT_2


title: Parental NCI-H358 cell line sensitive to sotorasib, rep1

value: sensitive to sotorasib


title: RNA-seq, hag704, root tips, rep2

city: Wuhan

first: Xiaoyuan

last: Guo

state: Hubei

address: No.1 Shizishan Street, Hongshan District

institute: Huazhong Agricultural University

description: Library name: LTSZ230

data_processing:
RNA-seq: Raw RNA-seq reads were quality-filtered using FastP (v 0.23.4) to remove adapter sequences and low-quality reads. Clean reads were aligned to the rice reference genome (MSU v7.0) using HISAT2 (v2.2.0). Differential gene expression analysis was performed with Cufflinks (v2.2.1). Genes with p value< 0.05 and fold change >2 (1.5 for acla2) in mutants relative to wild type were considered as differentially expressed genes.The root tip specifically expressed genes were identified by generating cuffnorm data by comparing root tips with other tissues. The FPKM (Fragments Per Kilobase of transcript per Million mapped reads) value of gene expression in root tips must be more than five times the average FPKM value of the same gene in other tissues. Additionally, the FPKM value in root tips must be at least 2 units higher than the maximum FPKM value observed in any of the other tissues. The enrichment of root specifically expressed genes compared to the genomic background was calculated by Fisher’s exactly test. CUT&Tag: Raw reads were filtered using FastP (v0.23.4) to eliminate adapter sequences and low-quality reads. Clean reads were aligned to the rice reference genome (MSU v7.0) using Bowtie2 (v2.5.2). Then, duplicated reads were discarded using samtools (v1.9). SICER2 software (version 1.0.2) was used to call histone modification peaks with “-w 200 -g 600 -f 150 -egf 0.74 -fdr 0.01” parameters. BigWig files generated by deepTools software with RPKM normalization were used for data visualization by IGV (version 2.3.88).
Assembly: RGAP version 7.0
Supplementary files format and content: RNA-seq:The diff file containing information of differential expression analysis between transcriptomes; CUT&Tag:The bigwig file incorporating data on chromosomal regions, signal intensity, and coverage.

organism: Oryza sativa Japonica Group

source_name: root

growth_protocol: In this study, the rice variety Zhonghua11 (Oryza sativa ssp. Japonica) was used to produce the transgenic plants. For in vitro cultures, seeds were surface-sterilized and germinated in media containing 0.3% phytagel supplemented with 2% (w/v) sucrose at 28 °C (in light) and 24 °C (in dark) with a 14 h light/10 h dark cycle.

extract_protocol:
RNA-seq: Total RNA was isolated from either 1 cm rice root tips of 7-day-old post-germination seedlings and 0.1 cm inflorescence meristems using the RNeasy Plus Micro Kit (Qiagen, 74034). First-strand cDNA was synthesized from 100 ng of total RNA using a reverse transcription kit (Vazyme, N712). Sequencing libraries were then prepared with the Vazyme TD502 kit and sequenced on an Illumina HiSeq 3000 system. CUT&Tag: Root tips from 7-day-old germinated rice seedlings were harvested to isolate nuclei. The nuclei were washed with wash buffer (Vazyme, TD903), followed by incubation with ConA Beads (Vazyme, TD903) at room temperature for 10 minutes.CUT&Tag experiment and DNA libraries were performed according to the instruction of Hyperactive Universal CUT&Tag Assay Kit (Vazyme, TD903).The PCR-amplified library was purified using VAHTS DNA Clean Beads (Vazyme, N411-01) and sequenced on the Illumina NovaSeq X Plus  platform.
RNA libraries for RNA-seq were prepared using TruePrep DNA Library Prep Kit V2 for Illumina following manufacturer's protocols. DNA libraries for CUT&Tag were prepared using Hyperactive Universal CUT&Tag Assay Kit for Illumina following manufacturer's protocols.

value: root

value: root tips


title: infect_day5_IRF3_3

city: Aarhus

first: Soren

last: Paludan

address: CF Møllers Alle 6, Building 1115

department: Biomedicine

country: Denmark

institute: Aarhus university

description: Library name: sample 15

data_processing:
Reads obtained from the DNBSEQ-G400 were aligned to a composite reference genome containing the mouse genome and HSV-1 genome using Cell Ranger v7.1 by 10x Genomics.
Assembly: GRCm39
Supplementary files format and content: Tab-separated count matrix, feature table, and barcode table

source_name: Brain stem

extract_protocol:
Animals were sedated with ketamine and xylazine and perfused with a minimum of 25 mL of PBS. 2-3 brain stems were pooled as one sample. Brain stems were cut into pieces and digested for 40 min at 37 °C with 5% CO2 in PBS supplemented with 1mg/mL Collagenase 1 (Roche), 10U/mL Pulmozyme (Dornase alpha) and 3.423 mg/mL Trehalose (Sigma Aldrich). 20 μl of sterile EDTA was added to each sample and incubated at 37°C for another 5 min. Tissues were then mechanically disrupted through a 70 μm cell strainer into a single-cell suspension.
Single-cell suspensions were washed with 25 mL 2% FCS-HBSS (Thermo Fisher Scientific), centrifuged, and resuspended in a mixture of PBS with 0.5% BSA (VWR) and Debris Removal Solution (Miltenyi Biotec). After overlaying PBS with 0.5% BSA, suspensions were centrifuged, resuspended, and adjust the cell count to 106 cells/mL. Single cell suspensions were converted to barcoded scRNAseq libraries following the manufacturer’s instructions. Quality control of libraries were performed with Tapestation 4200. After QC.

value: Brain stem

value: IRF3 knockout

value: HSV-1 infected


title: Parental NCI-H358 cell line sensitive to sotorasib, rep3


title: RNA-seq, hag704, root tips, rep1

description: Library name: LTSZ229


title: EpH4 WT_1

value: WT


title: infect_day5_IRF3_2

description: Library name: sample 14


title: uninfected_IRF3_1

description: Library name: sample 11

value: Control


title: DLD-1_DRB_10minR_rep1

city: Copenhagen

first: Haiyue

address: Blegdamsvej 3B

department: Department of Cellular and Molecular Medicine

institute: University of Copenhagen

description: Library name: sample_3

data_processing:
UMI sequences in R2 were extracted and attached to the read headers of R1 using UMI_tools v1.1.4. Sequencing adaptors in the UMI attched single-end reads (R1) were trimmed and low quality sequences were removed using trimgalore v0.6.6.
Reads were aligned to a human and yeast hybrid reference genome using STAR 2.7.9a with options:--outSAMattributes NH HI AS NM MD XS --outSAMstrandField intronMotif --outSAMmultNmax 1. Uniquely mapped reads were extracted from the alignments using samtools v1.15.1 with options: -q 255 -F 0x2. PCR duplicates were removed from the uniquely mapped reads using UMI_tools v1.1.4. The reads were further cleaned up by removing those mapped to exon-exon junctions using samtools v1.15.1 with options: -he '[XS]'.
Reads containing genes originated from the forward and reverse strands were split using samtools v1.15.1. Uniquely assigned features (i.e. genes)  were counted using featureCounts from Subread v2.0.3. Yeast spike-in size factors were calculated using DESeq2.  The bigwig files were generated from the forward and reverse strand mapped reads with normalization to yeast spike-in factors using  UCSC GenomeToolset and bedtools.
RNAPII transcription elongation rates for individual genes were calculated from the forward and reverse strands splitted reads using self-developed script as described here: https://github.com/haiyueliu/elongation_rate.
Assembly: GRCh38, Saccharomyces_cerevisiae.R64-1-1
Supplementary files format and content: bigwig files displaying read coverage profiles that can be viewed in a genome browser or IGV

extract_protocol:
Total RNA was harvested using TRIzol including an additional chloroform cleanup and isopropanol precipitated.
20  ug of total RNA were spiked in with 0.2 ug of yeast spike-ins (Saccharomyces cerevisiae W303a cells labeled with 5mM 4-thiouracil (4tU, Sigma, 440736) for 5 min, see Gregersen et al., 2022 for detailed procedure). RNA was fragmented for 20 min on ice by base hydrolysis and returned to a neutral pH buffer prior to biotinylation as previously described (Gregersen et al., 2020a). For addition of biotin to 4sU residues we used the MTSEA Biotin-XX linker (Biotium, BT90066). Biotinylated RNA was purified by phenol:chloroform and isopropanol precipitation, heat denatured and used for purification with uMACS Streptavidin microbeads (Miltenyi, 130-074-101). 4sU-RNA was eluted from beads using 2 rounds of 100 mM DTT and cleaned-up with the RNeasy MinElute Cleanup Kit (Qiagen, 74204) using 1.5x excess of ethanol for the RNA precipitation compared to the supplied protocol to ensure recovery of small RNA fragments. Concentration of 4sU-RNA was measured by Qubit (RNA high sensitivity assay, ThermoFisher Q32852) and the size range determined by high sensitivity RNA assay on a TapeStation (high sensitivity RNA ScreenTape, Agilent, 5067-5579). 4 ng of 4sU RNA was used for next-generation sequencing library preparation with the NEBNext Ultra II directional RNA kit (NEB, E7760) using Unique Dual Index UMI Adaptors (NEB, E7416) following the protocol for degraded RNA (RIN 1-2) without additional RNA fragmentation. Libraries were amplified using 12 PCR cycles, concentration measured by Qubit (DNA high sensitivity assay) and size distribution assessed using TapeStation (high sensitivity D5000 ScreenTape, Agilent, 5067-5592). The final library size distribution peaked at 390 nt, corresponding to an insert size of 260 nt. Libraries were sequenced on two P3 v100 flow cells (130 cycles) in paired-end mode on a NextSeq2000 (Illumina).

treatment_protocol: DRB/TTchem-seq2 was developed based on the previous protocol from (Gregersen et al, 2020a) with the major modifications being (1) the RNA labelling time, (2) shorter DRB release time points (3) inclusion of UMI adapters and (4) addition of a no DRB released sample for background subtraction.  For RNAPII synchronization close to the TSS USO2 cells were incubated with 100 uM DRB (5,6-dichlorobenzimidazole 1-β-D-ribofuranoside; Sigma-Aldrich, D1916) for 3.5 hrs and DRB inhibition released for 5, 10, 15 and 40 min by 3 washes pre-warmed PBS. For each time point newly synthesized RNA was labeled with 1 mM 4-thiourudine (4sU, Biosynth Carbosynth, NT06186) for 5 min immediately prior to harvest by addition of TRIzol (Thermo Fisher, 15596026) directly on top of the cell monolayer. Samples were prepared in duplicates for all time points.

value: Epithelial cell

value: DRB 3.5 hours, DRB release 10 minutes and 4sU labeling for the last 5 minutes


title: DLD-1_DRB_15minR_rep1

description: Library name: sample_4

value: DRB 3.5 hours, DRB release 15 minutes and 4sU labeling for the last 5 minutes


title: DLD-1_DRB_20minR_rep1

description: Library name: sample_5

value: DRB 3.5 hours, DRB release 20 minutes and 4sU labeling for the last 5 minutes


title: cmExoaCD11b

description: Library name: sampleB

value: Pancreatic cancer


title: NCC d5 HSTE rep10

city: London

first: Marco

last: Trizzino

state: UK

address: Imperial College Road

department: Life Sciences

country: United Kingdom

institute: Imperial College London

description:
Library name: NCC d5 HSTE rep10
ChiP-seq

library_source: genomic

data_processing:
ChIP-seq data were aligned to hg38 using BWA-mem. MACS2 was used to call peaks using default parameters and FDR <5%
Assembly: hg38 (homo sapiens)
Supplementary files format and content: Bigwig files

molecule: genomic DNA

growth_protocol: Human iPSCs were plated on Geltrex LDEV-Free hESC-qualified Reduced Growth Factor Basement Membrane Matrix and maintained in feeder-free, serum-free mTeSR Plus medium.

extract_protocol:
10 million hiPSC-NCCs were crosslinked in 1% paraformaldehyde for 5 min at RT with mild rotation. Crosslinking was quenched with 0.125M glycine by rotation for 5 min at RT. Cells were washed twice with cold PBS and were snap-frozen for 15 min on dry ice. Samples were stored at -80 °C until further processing. Cells were resuspended in 1 mL ChIP buffer (150 mM NaCl, 1% Triton X-100, 500 mM DTT, 10 mM Tris-HCl, 5 mM EDTA) supplemented with Pierce protease inhibitor tablet and incubated on ice for 10 min. SDS was added to each sample to a final concentration of 0.3%, and chromatin was sheared using the following settings: 9 min, duty factor 5%, 6°C in a Covaris M220 Focused-Ultrasonicator. Chromatin fragment size was assessed on a Tapestation 2200. The chromatin lysates were then centrifuged at 13,000 g for 10 min at 4 °C and supernatant was subsequently incubated with ChIP buffer supplemented with Pierce protease inhibitor and Dynabeads Protein G magnetic beads (Invitrogen) along with 3 μg of H3K9me3 antibody (Abcam, ab8898) and incubated overnight at 4 °C under mild rotation. The following day, samples were placed in a magnetic rack and washed twice with Mixed Micelle Wash Buffer (150 mM NaCl, 1% Triton X-100, 0.2% SDS, 20 mM Tris-HCl, 5 mM EDTA, 65% sucrose), twice with Buffer 200 (200 mM NaCl, 1% Triton X-100, 0.1% sodium deoxycholate, 25 mM HEPES, 10 mM Tris-HCl, 1 mM EDTA), twice with LiCl/detergent wash (250 mM LiCl, 0.5% sodium deoxycholate, 0.5% NP-40, 10 mM Tris-HCl, 1 mM EDTA), once with cold TE. The beads were resuspended in TE + 1% SDS and incubated at 65 °C for 10 min at 1200 rpm to elute immunocomplexes. The elution was repeated twice and the samples were incubated overnight at 65˚C to reverse cross-linking. The following day, Proteinase K (0.5 mg/mL) was added to digest samples at 65 °C for 1h. DNA was purified using the Zymo ChIP DNA Clean and Concentrator kit (Zymo, D5205) and quantified with the Quantifluor ONE dsDNA system (Promega, E4871).
Barcoded libraries were made with NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB, E7645L) using NEBNext Multiplex Oligos Dual Index Primers for Illumina (NEB, E7600S), and sequenced on Illumina NextSeq2000, producing 150 bp paired-end reads

treatment_protocol: For the generation of NCCs, the differentiation method was derived from the protocol published by Leung et al. (2016). Briefly, hiPSCs were grown in mTeSR Plus (StemCell Technologies, 100-0276) on Geltrex-coated (Thermofisher, A1413302) 6-well plates until reaching ~80% confluency. Differentiation was initiated by plating 2x106 hiPSCs in Neural Crest Differentiation media (DMEM/F12 (Gibco, A4192002), 2% B-27 supplement (Fisher Scientific, 15717988), 3 μM CHIR99021 (Stratech, S1263-SEL-5mg), 0.5% bovine serum albumin (Fisher Scientific, 12881630), 1X glutaMAX supplement (Gibco, 35050061), 1% penicillin-streptomycin (Gibco, 15070063)) onto Geltrex-coated 6-well plates supplemented with 10 μM Y-27632 (Cambridge Bioscience, HY-119937-5MG). The following days daily media changes were performed and 2 μg/mL doxycycline was added to the culture media from day 2 onwards (i.e. 24h after the start of differentiation). NCC differentiation was assessed after 5 days.

tag: chip antibody

value: Anti-trimethyl-Histone H3 (Lys4) Antibody, clone MC315, rabbit monoclonal (Merck #04-475)

value: hiPSC-derived cranial neural crest cells

value: plus gRNA

tag: protocol

value: cranial neural crest cell specification


title: DLD-1_DRB_5minR_rep1

description: Library name: sample_2

value: DRB 3.5 hours, DRB release 5 minutes and 4sU labeling at the same time for 5 minutes


title: EpH4 Cldn-null_3

value: mouse mammary gland epithelial cells

value: Cldn-null

value: Day 7


title: infect_day5_R278Q_2

description: Library name: sample 9

value: Brain stem

value: IRF3 R278Q mutation


title: infect_day5_R278Q_1

description: Library name: sample 8

value: HSV-1 infected


title: DLD-1_DRB_noR_rep1

description: Library name: sample_1

value: WT

value: DRB 3.5 hours, and 4sU labeling for the last 5 minutes


title: NCC d5 NOG rep10

description:
Library name: NCC d5 NOG rep10
ChiP-seq

value: minus gRNA


title: Parental NCI-H358 cell line sensitive to sotorasib, rep2

value: sensitive to sotorasib

value: no treatment


title: enriched nuclei_ZD_42

city: Cambridge

first: Giuliano

middle: Giuseppe

last: Stirparo

state: Cambridgeshire

address: 418 Cambridge Science Park Milton Rd

institute: Cerevance ltd

data_processing:
FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) was used for initial quality control. Adapter trimming was performed with Cutadapt v1.12 (Dobin et al., 2013). Reads were aligned to the hg38 human genome build (NCBI primary analysis set) using STAR aligner v2.5.2b (Wang et al., 2012) with splice junction files generated from Gencode release 27 and RefSeq GRCh38.p10. Post-alignment quality control was performed by examination of the STAR alignment metrics, Picard tools v2.8.2 (CollectRNASeqMetrics) (https://broadinstitute.github.io/picard/) and RSeQC v2.6.4 (tin.py) (Anders et al., 2015). Expression quantitation was performed with htseq-count v0.6.1p1 (Anders et al., 2015) using a custom annotation file based on RefSeq where both exonic and intronic reads are counted for each gene.  The read counts table from each sample was imported into RStudio (version 1.1.423; R version 3.4.3) running under CentOS Linux 7. Using edgeR (version 3.20.9), the read counts were normalized into Counts Per Million (TPM), log transformed, and batch corrected. For each individual cell type, we removed technical variance associated with batch effect using sva R package. Hierarchal clustering analysis was performed using the pheatmap R package (Kolde R et al., 2018). Principal component analysis was performed using pcaMethods R package. Metabolic flux analysis was computed using scFEA R package (https://github.com/changwn/scFEA, Alghamdi N et al., 2021). Cell type specific genes were identified using the specificity index algorithm previous described in (Xu et al., 2018) and (Dougherty et al., 2010). log2(TPM) values were used as gene expression input and ranks were averaged across 1,000 iterations.
Assembly: Gencode release 27 and RefSeq GRCh38.p10
Supplementary files format and content: fastq file containing raw data
Supplementary files format and content: txt files with raw counts

molecule: nuclear RNA

source_name: Cerebellum

extract_protocol:
RNA extractions from sorted nuclei were carried out with FFPE RNA Purification kits (Norgen: Product # 25300) using the manufacturer’s protocol from Step 2 (Lysate Preparation) with the following modifications: The On-column DNA removal Protocol was omitted. RNA binding and Column Wash centrifuge steps were carried out at 1,789×g for 2 min, followed by 1,789×g for 5 min to dry the resin. Elution was carried out in two steps, with 25 µL Elution Solution A added to each column followed by a 1 min room temperature incubation and centrifugation at 200×g for 2 min, then a further 15 µL Elution Solution A was added to each column with centrifugation at 1,789×g for 5 min. For samples containing >2,000 nuclei, the final 30 µL elution was split into two 15 µL aliquots. Purified RNA was vacuum dried in a CentriVap Benchtop Vacuum Concentrator (Labconco) for 40 min (or until no liquid was visible) at 4˚C and either used directly for library preparation or stored at -20˚C.
Sequencing library generation was carried out using the Trio RNA-Seq, Human rRNA Library Preparation Kit (Tecan: Product # 0506-A01) using the manufacturer’s protocol with the following modifications: Lyophilized RNA was resuspended in 12.5 µL DNAse mix and incubated at 37˚C for 30 min followed by 60˚C for 5 min and 4˚C Hold. Library Amplification I was carried out as 72˚C 2 min/95˚C 2 min/2×(95˚C 30 s/ 60˚C 60 s)/6×(95˚C 30 s/ 65˚C 60 s)/65˚C 5 min/4˚C Hold Library Amplification II was carried out as 95˚C 2 min/2×(95˚C 30 s/ 60˚C 60 s)/6×(95˚C 30 s/ 65˚C 60 s)/65˚C 5 min/4˚C Hold Final Libraries eluted in 30 µL DNA Resuspension Buffer (DR1) were checked on Agilent Bioanalyzer DNA 1000 chips, then measured on a Nanodrop 8000 and 32 sample pools (normalized to 500 ng/sample then diluted to 5 ng/µL) were sent for sequencing on an Illumina NextSeq High Output platform.

value: Cerebellum


title: uninfected_R278Q_2

description: Library name: sample 7

value: Control


title: uninfected_R278Q_1

description: Library name: sample 6


title: enriched nuclei_ZD_171

value: OPC


title: infect_day5_IRF3_1

description: Library name: sample 13

value: IRF3 knockout


title: enriched nuclei_UM_671


title: uninfected_IRF3_2

description: Library name: sample 12


title: infect_day5_WT_3

description: Library name: sample 5

value: Wilde type


title: enriched nuclei_UM_1508


title: infect_day5_WT_1

description: Library name: sample 3


title: enriched nuclei_UM_670

value: Oligo


title: enriched nuclei_UI_665


title: uninfected_WT_2

description: Library name: sample 2


title: enriched nuclei_UI_664


title: uninfected_WT_1

description: Library name: sample 1


title: Homo_2

city: Nanjing

first: Zhenzhen

address: Guangzhou Road#72

institute: Children’s Hospital of Nanjing Medical University

data_processing:
The sequencing data was filtered with SOAPnuke (v1.5.2) by (1) Removing reads containing sequencing adapter; (2) Removing reads whose low-quality base ratio (base quality less than or equal to 5) is more than 20%; (3) Removing reads whose unknown base ('N' base) ratio is more than 5%, afterwards clean reads were obtained and stored in FASTQ format.
The clean reads were mapped to the reference genome using HISAT2 (v2.0.4). Bowtie2 (v2.2.5) was applied to align the clean reads to the reference coding gene set then expression level of gene was calculated by RSEM (v1.2.12)
Assembly: mm9
Supplementary files format and content: TPM, FPKM, Readcount values for each Sample

source_name: liver

extract_protocol:
Liver tissues were harvested and rapidly frozen in liquid nitrogen. Total RNA was extracted from the tissues using Trizol (Invitrogen) according to the instruction manual.
Total RNA was extracted from the tissues using Trizol (Invitrogen, Carlsbad, CA, USA) according to manual instruction. About 100 mg of tissues were ground into powder by liquid nitrogen in a 2 mL tube, followed by being homogenized for 2 minutes and rested horizontally for 5 minutes. The mix was centrifuged for 5 minutes at 12,000×g at 4°C, then the supernatant was transferred into a new EP tube with 0.3 mL chloroform/isoamyl alcohol (24:1). The mix was shacked vigorously for 15s, and then centrifuged at 12,000×g for 10 minutes at 4°C. After centrifugation, the upper aqueous phase where RNA remained was transferred into a new tube with equal volume of supernatant of isopropyl alcohol, then centrifuged at 13,600 rpm for 20 minutes at 4°C. After deserting the supernatant, the RNA pellet was washed twice with 1 mL 75% ethanol, then the mix was centrifuged at 13,600 rpm for 3 minutes at 4°C to collect residual ethanol, followed by the pellet air dry for 5-10 minutes in the biosafety cabinet. Finally, 25μL~100μL of DEPC-treated water was added to dissolve the RNA. Subsequently, total RNA was qualified and quantified using a Nano Drop and Agilent 2100 bioanalyzer (Thermo Fisher Scientific, MA, USA).

value: liver

value: NEK7 hepatocyte knock-out


title: enriched nuclei_UI_1521


title: Homo_1


title: Homo_3


title: enriched nuclei_UD_660


title: enriched nuclei_UD_661


title: Ctrl_3


title: enriched nuclei_UD_659


title: enriched nuclei_ZD_79


title: Ctrl_1


title: enriched nuclei_UD_658


title: Ctrl_2


title: infect_day5_R278Q_3

description: Library name: sample 10


title: enriched nuclei_RS_656


title: NKO2

city: Shanghai

first: Wei

last: Yusheng

state: New York

address: 227,South Road of Chongqian,Shanghai,China

institute: Shanghai Jiao Tong University of Medicine

data_processing:
The sequencing data was filtered with SOAPnuke (v1.5.2)
The clean reads were mapped to the GRCm38 using HISAT2 (v2.0.4).
differential expression analysis was performed using the DESeq2(v1.4.5) with Q value ≤ 0.05.
Assembly: GRCm38 NCBI
Supplementary files format and content: text files include read count for each Sample

source_name: Bone marrow

extract_protocol:
RNA was harvested using Trizol reagent. 1 ug of total RNA for the construction of sequencing libraries.
RNA libraries were prepared for sequencing using standard protocols

value: Bone marrow

value: N-myc-induced B-ALL cells


title: enriched nuclei_RS_655


title: NKO1


title: enriched nuclei_RS_663


title: NWT2


title: infect_day5_WT_2

description: Library name: sample 4

value: Brain stem


title: enriched nuclei_RS_654


title: NWT1


title: enriched nuclei_RK_627


title: CAL62 cells,Erianin 50nM,48h,rep3

city: Tianjin

first: Minghan

last: Qiu

state: Tianjin

address: Department of Oncology, Tianjin Union Medical Center of Nankai Univercity, 190 Jieyuan Street, Hongqiao District, Tianjin, China.

department: Department of Oncology

description:
Library name: Sample6
CAL62-50E-3

data_processing:
Raw reads were filtered and cleaned by fastp software (v0.23.2).
Gene expression levels were quantified with featureCounts v2.0.1,referencing the GENCODE28 annotations.
Sequences were then aligned to the GRCh38 reference genome using STAR v2.7.10.
Transcript assembly and abundance estimation were performed using StringTie v2.2.1.
R was used for visualization.
Assembly: hg38
Supplementary files format and content: table-delimited text file includes raw counts for each sample
Supplementary files format and content: table-delimited text file includes TPM values for each sample

growth_protocol: CAL62 cells were maintained  in RPMI 1640, all with 10% FBS, penicillin (100 IU/ml), and streptomycin (100 μg/ml) in a 5% CO2 atmosphere at 37°C.

extract_protocol:
Total RNA was extracted using TRIzol reagent
RNA libraries were prepared using standard Illumina protocols.

treatment_protocol: CAL62 cells were treated with 50nM Erianin for 48h, with an equal concentration of DMSO as the control

value: ATC cell

value: Erianin 50nM, 48h


title: enriched nuclei_RK_623


title: CAL62 cells,Erianin 50nM,48h,rep1

institute: Tianjin Union Medical Center of Nankai Univercity

description:
Library name: Sample4
CAL62-50E-1

value: WT


title: enriched nuclei_RK_1498


title: enriched nuclei_QJ_653


title: enriched nuclei_QJ_650


title: CAL62 cells,DMSO,48h,rep3

description:
Library name: Sample3
CAL62-0E-3


title: Panc-1 negative control Rep B IgG

city: Udine

first: Raffaella

last: Picco

address: P.le Kolbe 4

department: Department of Medicine

country: Italy

institute: University of Udine

description:
Library name: G88
Reference for rG4 enrichment negative control

data_processing:
Basecalls performed using the FastQC/MultiQC programs
Raw reads were clipped from their adapter sequences using the Trimmomatic software (v0.35). Reads with an average Phred quality lower than 28 and length < 36 bp were discarded
ChIP-seq reads were aligned to the hg38 genome assembly using bowtie2 version version 2.3.5.1 with default configuration for paired reads
SAM files filtered using samtools to filter out reads with mapping quality < 10
Duplicate reads were flagged using samtools but no duplicates were removed
Peaks were called using MACS2 version 2.2.9.1  in its paired-end  analysis mode ('-f BAMPE')
Assembly: GRCh38 (Ensembl 107)
Supplementary files format and content: Coverage tracks were generated using the bamCoverage (deepTools) tool, taking an alignment of reads as input (BAM file) and generating a coverage track (bigWig) as output, applying the following parameters: --outFileFormat bigwig --binSize 50 -p 10 --normalizeUsing RPKM
Supplementary files format and content: BigWig, narrowPeak (except for Input samples)

source_name: Pancreatic ductal adenocarcinoma

growth_protocol: Cells were grown in DMEM (Euroclone, Italy) supplemented with 10 % FBS, penicillin/streptomycin and 2 mM L-glutamine (Euroclone, Italy) at 37 °C, 5 % CO2. 1µM Pyridostatin was used where indicated (PDS SML2690, Sigma-Aldrich, Germany),

extract_protocol:
30 x 10^6 Panc-1 cells, treated or not for 16 h with 1 µM PDS, were collected and then lysed for 10 min. with Hypotonic Lysis buffer (5 mM Pipes, 85 mM KCl, 0.5 % NP40) containing protease inhibitor cocktail. Pellets containing nuclei were discarded while cytoplasmic fraction was diluted 1:1 in G4RP buffer (150 mM KCl, 25 mM Tris pH 7.4, 5 mM EDTA, 0.5 mM DTT and 0.5 % NP40 in nuclease-free water). After preclearing, lysates were incubated with  3 μg of BG4 (Merck) antibody and 8 μl Dynabeads Protein G for 6h at 4°C. After washings, beads were incubated with 1ml of Tri-Reagent (Sigma-Aldrich, Germany) to extract RNA with a phenol/chloroform extraction method. RNA was DNAseI (Thermo Fisher Scientific) treated to digest genomic DNA. Concentration, integrity and purity of RNA were checked using Agilent 2100 Bioanalyzer (Agilent RNA 6000 Nano Kit).
10 ng of RNA was digested with DNAseI (NEB), freed from rRNA (using the ribo-zero method) and reverse transcribed to obtain cDNA. Adapters were ligated to the fragmented cDNA after end repair and A-tailing according to  Illumina protocols. 16 rounds of PCR amplification were performed to enrich the cDNA fragments. The PCR products were then purified using Ampure XP beads (Agencourt) and subjected to paired-end 100 sequencing at the Genewiz Genomics facility (Genewiz, Germany).

value: Pancreatic ductal adenocarcinoma

value: PDAC

value: diploid


title: CAL62 cells,DMSO,48h,rep1

description:
Library name: Sample1
CAL62-0E-1


title: enriched nuclei_PW_648

value: Cerebellum


title: enriched nuclei_PW_646


title: CAL62 cells,DMSO,48h,rep2

description:
Library name: Sample2
CAL62-0E-2


title: enriched nuclei_QJ_1505


title: WT-T1

city: shanghai

first: Xu

last: Chu

address: Dong'an Road

institute: Fudan University Shanghai Cancer Center

description: Library name: Sample 6

data_processing:
library strategy: Target Transcript Amplification and Sequencing (TATA-seq)
Reads were trimmed using Cutadapt
mapping mapped to hg38  use STAR
peak Calling use MACS3
Assembly: hg38
Supplementary files format and content: bigwig

molecule: cytoplasmic RNA

source_name: HeLa

growth_protocol: HeLa cells cultured in DMEM high glucose medium (BasalMedia) supplemented with 10% fetal bovine serum (VivaCell) and 1% penicillin-streptomycin (Gibco) at 37°C with 5% CO2 in the humidified incubator.

extract_protocol:
For sodium arsenite treatment, HeLa cells with 80% confluence were cultured in prewarmed medium containing 0.5 mM of sodium arsenite (Sigma-Aldrich) under culture conditions for 1 h. The cells that only changed the medium simultaneously were set as the control group.
Fixed cells by 4% PFA for 15 min. After washed with PBS and added permeabilization buffer  and incubated on ice for 15 min. Washed the cells and blocked with blocking buffer at room temperature for 1h. Diluted the antibody of G3BP1 (Abcam, ab181150; 1:200 dilution) in antibody buffer. The concentration of the IgG antibody (Abclonal, AC005) should be the same as the primary antibody. Next, cells were incubated with antibodies on the rotation shaker at 25°C for 2 h. Washed cells with primary antibody wash buffer and PBST (PBS with 0.1% Tween-20) for three times respectively.The guinea pig anti-rabbit IgG (heavy & light chain) antibody (Antibodies Online, ABIN101961) was labeled with streptavidin through the Streptavidin Conjugation Kit (Abcam, ab102921). Subsequently, added 12.5 μM biotinylated T7-N9 oligo-M-EB (1:100 dilution; sequence 5'Bio-TCACTGTAGTTGTACAGACTCCATAATACGACTCACTATAGGGATAATGAGCAGACGTGTGCTCTTCCGATCT-NNNNNNNNN-3') and the streptavidin-secondary antibody (1:150 dilution) into the low adhesion tube and incubated at 4°C for 20 min on the rotation shaker to assemble the streptavidin-antibody-biotinylated-oligo triplex. Purified the triplex by the Amicon® Ultra Centrifugal Filter 100 kDa MWCO (Millipore) to remove the excessive biotinylated oligo. Dissolved the triplex with primary antibody wash buffer supplemented with 3% BSA and incubate the cells on the shaker at 25 °C for 45-60 min. Washed the cells with primary antibody wash buffer, wash buffer I, wash buffer II and PBST three times, respectively. Then rinsed the well with PBS twice times, incubated with PBS at 37°C for 10 min, ramped to 25°C for 5-10 min and hold at 25°C. The in situ reverse transcription was performed by SuperScript™ III (Thermo Fisher Scientific) at 37°C for 1 h. Washed the cells with 10 mM Tris HCl (pH 7.5), performed second strand synthesis by DNA Polymerase I (E. coli) (NEB), E. coli DNA Ligase (NEB) and RNase H (NEB). Added T4 DNA polymerase (NEB) and incubate at 16°C for 15 min for end repair. Then cells were digested with Proteinase K solution (Thermo Fisher Scientific) at 55°C for 30 min. Purified the dsDNA product by DNA Clean & Concentrator-5 Kit (Zymo research). Next, transcript and linear amplificated with T7 RNA Polymerase (Vazyme) at 37°C for 2-4 h and purified RNA by Dynabeads MyOne Silane (Thermo Fisher Scientific). Assembled the T4 Polynucleotide Kinase (NEB) reaction and incubated at 37°C for 30 min. After silane beads clean-up, performed the 3’ adapter (sequence 5'Phos-AGATCGGAAGAGCGTCGTG-3'Bio) ligation by T4 RNA Ligase 2 truncated KQ (NEB) at 25°C for 4 h or 16°C overnight. Then purified the RNA with silane beads. Reverse transcription (primer sequence 5'-ACACGACGCTCTTCCGATCT-3') was performed by M-MLV Reverse Transcriptase (Promega). Purified with silane beads and library amplification by NEBNext® Ultra™ II Q5® Master Mix (NEB). The library was purified by AMPure XP beads (Beckman) and 2% low melting agarose (Solarbio) gel with size selection between 200 and 500 bp. Purification cDNA from agarose gels through Gel DNA Recovery Kit (Zymo research) and sequencing.

value: HeLa

value: Human cervical cancer cell

value: wild type


title: WT-IgG

description: Library name: Sample 5


title: WT-T3

description: Library name: Sample 8


title: WT-T2

description: Library name: Sample 7


title: enriched nuclei_QJ_649


title: Panc-1 negative control Rep A IgG

description:
Library name: G85
Reference for rG4 enrichment negative control

treatment_protocol: 1µM Pyridostatin was used where indicated (PDS SML2690, Sigma-Aldrich, Germany),


title: KO-T2

description: Library name: Sample 3


title: CAL62 cells,Erianin 50nM,48h,rep2

description:
Library name: Sample5
CAL62-50E-2


title: enriched nuclei_PW_645


title: KO-T1

description: Library name: Sample 2


title: enriched nuclei_P58/10_3461

value: OPC


title: enriched nuclei_P24/17_3519


title: enriched nuclei_RK_625


title: KO-T3

description: Library name: Sample 4


title: enriched nuclei_P21/17_3480


title: enriched nuclei_P24/17_3518

value: Oligo


title: Ctrl-IgG-2

description: Library name: Sample 8

data_processing:
Reads were trimmed using Cutadapt
mapping mapped to hg38  use STAR
peak Calling use MACS3
Assembly: hg38
Supplementary files format and content: bigwig
Library strategy: TATA-seq


title: enriched nuclei_OY_684


title: Ctrl-IgG-1

description: Library name: Sample 7


title: Ctrl-G3BP-2

description: Library name: Sample 6


title: enriched nuclei_OY_681


title: enriched nuclei_P58/10_3462


title: enriched nuclei_OY_680


title: Ctrl-G3BP-1

description: Library name: Sample 5


title: AS-IgG-1

description: Library name: Sample 3


title: enriched nuclei_OY_3344


title: AS-G3BP-2

description: Library name: Sample 2


title: enriched nuclei_OY_10444

value: Microglia


title: enriched nuclei_NJ_706


title: AS-G3BP-1

description: Library name: Sample 1


title: enriched nuclei_NJ_705


title: Day07_EC_Cd31po_Rnf20_KD_rep2

city: Mannheim

first: Gergana

last: Dobreva

state: Baden Württemberg

address: Ludolf-Krehl-Str. 7-11

department: Cardiovascular Genomics and Epigenomics

institute: Medical Faculty Mannheim/University of Heidelberg

description: Library name: Day07_EC_Cd31po_Rnf20_KD_r2

data_processing:
High quality trimmed reads were mapped to the mm10 reference genome using STAR (v. 2.7.10a) (−alignIntronMin 20 −alignIntronMax 500000) (Dobin et al., 2013).
Mapped sam files were used to create makeTaglibraries using makeTagDirectory (default settings).
analyzeRepeats.pl function (rna mm10 -strand both -count genes –noadj) from HOMER (Heinz et al., 2010)  was used to quantify the mapped reads.
Differential expression was quantified and normalized by Deseq2 (v. 1.40.1). rpkm.default (defaults) from EdgeR (v. 3.42.4) was used to calculate the reads per kilobase of millon reads (RPKM).
Bam files were merged with bamtools merge (v. 2.5.1) (default settings) and converted to BigWig files with the help of bamCoverage (v. 3.5.1) settings (-bs 20 --smoothLength 40 -p max  --normalizeUsing RPKM -e 150)
Assembly: mm10 from UCSC genome
Supplementary files format and content: BigWig files
Supplementary files format and content: Excel file from differentialy expression analysis

source_name: EC derived from ESC

growth_protocol: E14 Tg(Nkx2-5-EmGFP) embryonic stem (ES) cells were differentiated into endothelial cells (ECs) following a modified version of an established method (https://doi.org:10.1016/j.stemcr.2017.07.001). Before differentiation, ES cells were cultured feeder-free for three days on gelatin-coated dishes in a priming medium consisting of a 1:1 mixture of Neurobasal Medium and DMEM/F-12, supplemented with 0.5X N2 Supplement, 0.5X B27 Supplement, 1X Penicillin/Streptomycin, 1X L-Glutamine, 0.05% Bovine Albumin Fraction V, 150 µM monothioglycerol, 10 ng/ml Bone Morphogenetic Protein 4 (BMP4), and 2000 U/ml Leukemia Inhibitory Factor (LIF). Differentiation was initiated by seeding 75,000 cells/ml into ultra-low attachment Petri dishes containing differentiation medium composed of 75% Iscove’s Modified Dulbecco’s Medium (IMDM) and 25% Ham’s F-12 Nutrient Mix, supplemented with 0.5X N2 Supplement, 0.5X B27 Supplement (without vitamin A), 1X Penicillin/Streptomycin, 50 µg/ml L-Ascorbic Acid, and 450 µM monothioglycerol. After 48 hours (Day 2), embryoid bodies (EBs) were harvested, washed with PBS, and enzymatically dissociated using Accutase. The resulting single-cell suspension was counted, and cells were re-aggregated at 150,000 cells/ml in a differentiation medium supplemented with vascular endothelial growth factor A (VEGF-A), Activin A, and BMP4. EC differentiation, cells from the mesodermal stage (Day 3.75) were seeded at 300,000 cells/cm² onto Matrigel coated dishes and cultured in Endothelial Cell Differentiation Medium, which consisted of a 1:1 mixture of IMDM and Ham’s F-12 Nutrient Mix, supplemented with 0.5X N2 Supplement, 0.5X B27 Supplement, 0.05% lipid-rich BSA, 50 ng/ml L-Ascorbic Acid, 50 ng/ml monothioglycerol, 50 ng/ml VEGF, 10 ng/ml bFGF, and 2 ng/ml BMP4. The medium was replaced every second day. By Day 7,  differentiated ECs were obtained.

extract_protocol:
Total RNA was isolated using QiaZOl RNA extraction kit from Qiagen
Hieff NGS C102P1 Ultima Dual-mode mRNA

treatment_protocol: At the mesodermal stage (D3.7), cells were transduced overnight with lentiviral supernatants carrying either control (pLKO1) or shRnf20 constructs. The viral supernatants were produced either in EC differentiation medium. The next morning, viral supernatants were removed, and fresh differetiation medium was added.

value: EC derived from ESC

value: EC

value: mouse epithelial cells

value: viral transfection with shRnf20


title: Day07_EC_Cd31po_CtrPlko_rep1

description: Library name: Day07_EC_Cd31po_CtrPlko_r1

value: Ctr

value: viral transfection with Empty Plko Vector


title: enriched nuclei_MD_6255_8341


title: KO-IgG

description: Library name: Sample 1


title: enriched nuclei_MD_6252_9079


title: enriched nuclei_MD_6255_5646


title: enriched nuclei_MD_6255_5641


title: Day07_EC_Cd31po_CtrPlko_rep2

description: Library name: Day07_EC_Cd31po_CtrPlko_r2


title: enriched nuclei_MD_6255_5574

value: Astrocyte


title: WT_AM_3

first: Jiaoyan

last: Lv

address: 30 Shuangqing Road, Haidian District

institute: Tsinghua University

data_processing:
The raw reads were filted with SOAPnuke v1.5.2 to remove adapter-containing and low quality reads
Clean reads were mapped to reference genome (GCF_000001635.26_GRCm38.p6, NCBI) using HISAT v2.0.4
Clean reads were mapped to reference gene sequence by Bowtie2 v2.2.5 to analysis gene expression
Assembly: GCF_000001635.26_GRCm38.p6, NCBI
Supplementary files format and content: excel file includes FPKM for each sample
Supplementary files format and content: excel file includes TPM for each sample

source_name: primary alveolar macrophages

extract_protocol:
Total RNA of isolated cells or lung tissue was extracted using TRIzol Reagent
RNA library was prepared with MGIEasay RNA Library Prep Set following manufacturer's protocol

value: primary alveolar macrophages

value: WT

value: allergen exposed


title: enriched nuclei_P21/17_3838

value: Cerebellum

value: Purkinje


title: KO_AM_3

value: Trim33 knockout


title: Day07_EC_Cd31po_Rnf20_KD_rep1

description: Library name: Day07_EC_Cd31po_Rnf20_KD_r1


title: enriched nuclei_MD_6252_9078


title: enriched nuclei_NJ_704


title: WT_AM_2


title: KO_AM_1


title: enriched nuclei_MD_6252_9077


title: enriched nuclei_MD_6252_6662


title: enriched nuclei_MD_6252_6652


title: enriched nuclei_OY_682


title: enriched nuclei_MD_6252_6640


title: enriched nuclei_MD_6252_6556


title: AS-IgG-2

description: Library name: Sample 4

value: HeLa

value: Human cervical cancer cell

value: wild type


title: enriched nuclei_MD_6252_6672


title: EMO6

city: fuzhou

first: qiyuan

last: Yang

state: Fujian Province

address: The First Affiliated Hospital of Fujian Medical University, Taijiang District, Fuzhou City, Fujian Province

description: Library name: sample14

data_processing:
Quality of bases lower than 20, pass filter data of fastq format were processed by Cutadapt (V1.9.1, phred cutoff: 20, error rate: 0.1, adapter overlap: 1bp, min. length: 75, proportion of N:0.1) properly. Then, with the file as a reference gene file, HTSeq (v0.6.1) estimated gene and isoform expression levels from the pair-end clean data.
GOSeq (v1.34.1) was used identifying Gene Ontology (GO) terms that annotate a list of enriched genes with a significant padj less or equal than 0.05. And topGO was used to plot DAG. Novel transcripts prediction After assembling a transcriptome from one or more samples, Cuffcompare, a tool of Cufflinks v2.2.1, compared assembly to known transcripts. Then, novel transcripts can be predicted via results of Cuffcompare.
Alternative splicing analysis rMATs v4.1.0 is a computational tool to detect differential alternative splicing events from RNA-Seq data. And resulting bam files of aligner HISAT2 had been used as input during the analysis. SNV analysis Samtools v0.1.19 with command mpileup and Bcftools v0.1.19 were used to do SNV calling.
Assembly: mm10
Supplementary files format and content: All_gene_counts.xls

growth_protocol: After purchase, the mice were allowed to acclimate for 7 days in the experimental animal facility. The ambient temperature was maintained at 22–24 °C with a 12-hour light/12-hour dark cycle, and they were given free access to water.

extract_protocol:
RNA was harvested using MagPure Universal RNA LQ Kit 2. 1 ug of total RNA was used for the construction of sequencing libraries.
RNA libraries for RNA-seq were prepared using VAHTS® Universal V8 RNA-seq Library Prep Kit  for Illumina following manufacturer's protocols.

treatment_protocol: Mice were intragastrically dosed with emodin (400 mg/kg) in 0.5% CMC-Na solution for three consecutive weeks. Control animals received 0.5% CMC-Na alone.  Liver samples were collected immediately after euthanasia following the final administration.

value: liver

value: emodin


title: EMO7

institute: The First Affiliated Hospital of Fujian Medical University

description: Library name: sample15


title: EMO8

description: Library name: sample16


title: KO_AM_2

city: Beijing


title: EMO5

description: Library name: sample13


title: WT_AM_1


title: enriched nuclei_MD_6247_6998


title: EMO3

description: Library name: sample11


title: EMO4

description: Library name: sample12


title: enriched nuclei_MD_6249_6187


title: enriched nuclei_MD_6209_6862

value: Microglia


title: WT6

description: Library name: sample6

value: control


title: WT7

description: Library name: sample7


title: enriched nuclei_MD_6209_6799


title: enriched nuclei_MD_6247_8349


title: EMO1

description: Library name: sample9


title: WT8

description: Library name: sample8


title: enriched nuclei_MD_6247_6738

value: Oligo


title: WT4

description: Library name: sample4


title: WT3

description: Library name: sample3


title: EMO2

description: Library name: sample10


title: enriched nuclei_MD_6209_6850


title: enriched nuclei_MD_6018_6715

value: OPC


title: enriched nuclei_MD_6209_6795


title: WT2

description: Library name: sample2


title: WT1

description: Library name: sample1


title: enriched nuclei_MD_6018_6680


title: enriched nuclei_MD_6018_6665


title: enriched nuclei_MD_6018_6671


title: BA-treated rep 4

city: Hangzhou

first: BIhui

last: Jin

address: 158#, Shangtang road

institute: zhejiang provincal people's hospital

description: Library name: B4

data_processing:
RNA-seq
fastp software (https://github.com/OpenGene/fastp) were used to remove the reads that contained adaptor contamination, low quality bases and undetermined bases with default parameter. Then sequence quality was also verified using fastp. We used Ensembl (ftp://ftp.ensembl.org/pub/release-v112/fasta/mus musculus/dna/) to map reads to the reference genome of mus musculus Ensembl_v112. The mapped reads of each sample were assembled using StringTie (https://ccb.jhu.edu/software/stringtie) with default parameters. Then, all transcriptomes from all samples were merged to reconstruct a comprehensive transcriptome using gffcompare (https://github.com/gpertea/gffcompare/). After the final transcriptome was generated, StringTie and was used to estimate the expression levels of all transcripts. StringTie was used to perform expression level for mRNAs by calculating FPKM (FPKM = [total_exon_fragments / mapped_reads(millions) × exon_length(kB)]). The differentially expressed mRNAs were selected with fold change > 2 or fold change < 0.5 and with parametric F-test comparing nested linear models (p value < 0.05) by R package edgeR (https://bioconductor.org/packages/release/bioc/html/edgeR.html).
Assembly: mus_musculus Ensembl_v112
Supplementary files format and content: tab-delimited text files include FPKM and count values for each sample

growth_protocol: Female C57BL/6J mice (2 weeks old) were housed in a specific pathogen-free (SPF) facility under controlled environmental conditions (temperature 22 ± 2 °C, relative humidity 50 ± 10%, and a 12-h light/dark cycle) with ad libitum access to standard chow and water. Mice were acclimated for 1 week prior to the start of treatments.

extract_protocol:
Total RNA was isolated and purified using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer's procedure.  The RNA amount and purity of each sample was quantified using NanoDrop ND-1000 (NanoDrop, Wilmington, DE, USA).  The RNA integrity was assessed by Bioanalyzer 2100 (Agilent, CA, USA) with RIN number >7.0, and confirmed by electrophoresis with denaturing agarose gel.
Poly (A) RNA is purified from 1μg total RNA using Dynabeads Oligo (dT)25-61005 (Thermo Fisher, CA, USA) using two rounds of purification.   Then the poly(A) RNA was fragmented into small pieces using Magnesium RNA Fragmentation Module (NEB, cat.  e6150, USA) under 94℃ 5-7min.   Then the cleaved RNA fragments were reverse-transcribed to create the cDNA by SuperScript™ II Reverse Transcriptase (Invitrogen, cat.   1896649, USA), which were next used to synthesise U-labeled second-stranded DNAs with E. coli DNA polymerase I (NEB, cat.  m0209, USA), RNase H (NEB, cat.  m0297, USA) and dUTP Solution (Thermo Fisher, cat.  R0133, USA）.   An A-base is then added to the blunt ends of each strand, preparing them for ligation to the indexed adapters.   Each adapter contains a T-base overhang for ligating the adapter to the A-tailed fragmented DNA.   Single- or dual-index adapters are ligated to the fragments, and size selection was performed with AMPureXP beads.   After the heat-labile UDG enzyme (NEB, cat.  m0280, USA) treatment of the U-labeled second-stranded DNAs, the ligated products are amplified with PCR by the following conditions: initial denaturation at 95℃ for 3 min;   8 cycles of denaturation at 98℃ for 15 sec, annealing at 60℃ for 15 sec, and extension at 72℃ for 30 sec;   and then final extension at 72℃ for 5 min. The average insert size for the final cDNA library was 300±50 bp.   At last, we performed the 2×150bp paired-end sequencing (PE150) on an illumina Novaseq™ 6000 (LC-Bio Technology CO., Ltd., Hangzhou, China) following the vendor's recommended protocol.

treatment_protocol: Female C57BL/6J mice (3 weeks old) were randomly assigned to three groups. Control group: Subcutaneous implantation of empty silicone tubes for 12 weeks. PCOS-NAFLD model group: Subcutaneous implantation of silicone tubes containing 10 mg dihydrotestosterone (DHT), replaced every 4 weeks, for a total of 12 weeks. BA treatment group: Same DHT implantation protocol as the model group, with additional oral administration of baicalin (200 mg/kg/day) starting from week 5 and continued for 8 weeks.

tag: Sex

value: Female


title: BA-treated rep 3

description: Library name: B3


title: enriched nuclei_MD_5902_5712


title: BA-treated rep 2

description: Library name: B2


title: enriched nuclei_MD_5902_5705


title: WT5

description: Library name: sample5


title: enriched nuclei_MD_6209_6849


title: enriched nuclei_MD_5902_5701

value: Astrocyte


title: PCOS-NAFLD rep 4

description: Library name: D4


title: enriched nuclei_MD_5751_5655


title: PCOS-NAFLD rep 3

description: Library name: D3


title: enriched nuclei_MD_5751_5656


title: PCOS-NAFLD rep 2

description: Library name: D2


title: enriched nuclei_MD_5751_5652


title: PCOS-NAFLD rep 1

description: Library name: D1


title: enriched nuclei_MD_5751_5653


title: enriched nuclei_MD_5751_5576


title: Control rep 4

description: Library name: C4


title: MI_F_InVivo

city: Los Angeles

first: Lili

state: California

address: 610 Charles E. Young Dr. East

department: Microbiology Immunology & Molecular Genetics

institute: University of California, Los Angeles

description:
Library name: Sample_07
10x Genomics

data_processing:
For cell clustering and annotation, the merged digital expression matrix generated by Cellranger was analyzed using an R package Seurat (v.5.3.0) following the guidelines. Briefly, after filtering the low-quality cells, the expression matrix was normalized using NormalizeData function, followed by selecting variable features across datasets using FindVariableFeatures and SelectIntegrationFeatures functions. To correct the batch effect, Harmony function were used based on the selected feature genes. The corrected dataset was subjected to standard Seurat workflow for dimension reduction and clustering. In this study, clusters of therapeutic cells were manually merged and annotated based on gene signatures reported from Human Protein Atlas (proteinatlas.org) and previous studies. AddModuleScore was used to calculate module scores of each list of gene signatures, and FeaturePlot function was used to visualize the expression of each signature in the UMAP plots.
Assembly: Human (GRCh38) 2024-A
Supplementary files format and content: matrix.mtx.gz: Matrix Market Format, with rows representing genes and columns representing cells.
Supplementary files format and content: barcode.tsv.gz: Tab-separated values, where each row represents a unique identifier for a cell.
Supplementary files format and content: features.tsv.gz: Tab-separated values, where each row represents a gene.

source_name: In vitro

extract_protocol:
Therapeutic cell products were subjected to FACS sorting for isolating live AlloMCAR-NKT cells.
Cells were quantified using a Cell Countess II automated cell counter (Invitrogen/Thermo Fisher Scientific). A total of 10,000 cells from each experimental group were loaded on the Chromium platform (10X Genomics), and libraries were constructed using the Chromium Next GEM Single Cell 3' Kit v3.1 and the Chromium Next GEM Chip G Single Cell Kit (10X Genomics), according to the manufacturer’s instructions. Library quality was assessed using the D1000 ScreenTape on a 4200 TapeStation System (Agilent Technologies). Libraries were sequenced on an Illumina NovaSeq using the NovaSeq S4 Reagent Kit (100 cycles; Illumina).

value: In vitro

value: Restimulation

value: NKT cell


title: Control rep 3

description: Library name: C3


title: enriched nuclei_MD_5730_8394

value: Cerebellum


title: enriched nuclei_MD_5751_5649


title: enriched nuclei_MD_5730_8331


title: Control rep 2

description: Library name: C2


title: enriched nuclei_MD_5751_5650


title: MI_C_peritonealCavity

description:
Library name: Sample_06
10x Genomics

source_name: Ascites

value: Ascites

value: Day 28


title: MI_B_spleen

description:
Library name: Sample_05
10x Genomics

source_name: Spleen

value: Spleen

value: Day 14


title: enriched nuclei_MD_5730_8327


title: MI_B_peritonealCavity

description:
Library name: Sample_04
10x Genomics


title: enriched nuclei_MD_5730_8329


title: Control rep 1

description: Library name: C1


title: enriched nuclei_MD_5902_5703


title: BA-treated rep 1

description: Library name: B1


title: enriched nuclei_MD_5730_7245


title: MI_B_liver

description:
Library name: Sample_03
10x Genomics

source_name: Liver

value: Liver


title: enriched nuclei_MD_5730_7244


title: rats underwent the same surgical procedure without MPG injury, Sham_3

city: Suzhou

first: Qiang

last: Shan

address: No. 100, Chongwen Road, Suzhou Industrial Park

institute: Suzhou Institute of Systems Medicine

description: Library name: Sham_3

data_processing:
Reads mapping: Reference genome and gene model annotation files were downloaded from genome website directly. Index of the reference genome was built using Hisat2 v2.0.5 and paired-end clean reads were aligned to the reference genome using Hisat2 v2.0.5. We selected Hisat2 as the mapping tool for that Hisat2 can generate a database of splice junctions based on the gene model annotation file and thus a better mapping result than other non-splice mapping tools.
Assembly: grcm38_p6
Supplementary files format and content: .xlsx

source_name: cell line

extract_protocol:
RNA extraction and detection Reagent kit: InvitrogenTRIzol reagent Operation steps: 1. Homogenization treatment Take the sample in a 2ml grinding tube and thoroughly grind it using a grinder at 60Hz for 60 seconds. Then take 2-4 samples each time, add 1ml TRIzol reagent to the ground powder and mix immediately (the sample needs to be kept at low temperature before adding TRIzol). After adding all the samples to TRIzol, wet beat at 55Hz for 30 seconds and incubate at room temperature for 5 minutes to facilitate complete separation of nuclear protein bodies in the homogenized sample. Centrifuge at 12000rpm for 5 minutes and take the supernatant (a 1.5ml centrifuge tube can be prepared during centrifugation to hold the supernatant). 2. Phase separation Add 0.2ml chloroform to the supernatant, shake the tube vigorously for 30 seconds, and incubate at room temperature for 2-3 minutes. Centrifuge at 4 ° C and 12000rpm for 15 minutes (during centrifugation, prepare a 1.5ml centrifuge tube and write the sample name and number). The mixture separates into a red lower layer (phenol chloroform phase), an intermediate phase, and an upper colorless aqueous phase. RNA exists in the aqueous phase. 3. RNA precipitation Transfer 400 μ l of aqueous phase to a 1.5ml final tube, add 500 μ l of isopropanol, invert and mix well (about 30 times). Incubate the sample at room temperature for 10 minutes. Centrifuge at 4 ° C and 12000rpm for 10 minutes. 4. RNA washing Discard the supernatant and add 1ml of 75% ethanol to wash the precipitate (try to resuspend the precipitate as much as possible). Centrifuge at 4 ° C and 12000rpm for 5 minutes. 5. Dissolve RNA again At the end of the process, briefly centrifuge and extract ethanol, then add an appropriate amount of RNase free water-soluble sample directly. Do not vacuum centrifuge RNA. It is important not to let the RNA precipitate completely dry, as this will greatly reduce its solubility.
Total RNA was used as input material for the RNA sample preparations. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in First Strand Synthesis Reaction Buffer(5X). First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase(RNase H-). Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3’ ends of DNA fragments, Adaptor with hairpin loop structure were ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 370~420 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system.

value: The penile tissue of rats

value: pluripotent stem cells


title: rats underwent the same surgical procedure without MPG injury, Sham_2

description: Library name: Sham_2


title: MI_B_blood

description:
Library name: Sample_02
10x Genomics

source_name: Blood

value: Blood


title: rats underwent the same surgical procedure without MPG injury, Sham_1

description: Library name: Sham_1


title: enriched nuclei_MD_5669_6748


title: enriched nuclei_MD_5669_7292

value: Purkinje


title: MI_A_InVivo

description:
Library name: Sample_01
10x Genomics

value: Pre-infusion


title: enriched nuclei_MD_5669_6745


title: DAC_SCC15

city: Shiyan

first: Qin

last: Shanshan

address: Renmin Road 30, Maojian District

institute: Institute of Basic Medical Sciences, School of Basic Medical Sciences, Hubei University of Medicine

description: Library name: Sample 4

data_processing:
Raw data (raw reads) of fastq format were firstly processed through in-house perl scripts. In this step, clean data (clean reads) were obtained by removing reads containing adapter, reads containing ploy-N and low quality reads from raw data. At the same time, Q20, Q30 and GC content the clean data were calculated. All the downstream analyses were based on the clean data with high quality.
Reads were mapped to human genome assembly GRCh38 (ftp://ftp.ensembl.org/pub/release-89/fasta/homo_sapiens/dna/Homo_sapiens.GRCh38.dna.toplevel.fa.gz) using HISAT2 with default parameters.
HTSeq v0.13.5 was used to count the reads numbers mapped to each gene. And then FPKM of each gene was calculated based on the length of the gene and reads count mapped to this gene. GTF file:ftp://ftp.ebi.ac.uk/pub/databases/gencode/Gencode_human/release_29/gencode.v29.annotation.gtf.gz.
Assembly: GRCh38
Supplementary files format and content: File include Count and FPKM values for each sample

growth_protocol: The human OSCC cell lines CAL27 and SCC15 were purchased from the Shanghai Cell Bank of Chinese Academy of Sciences (Shanghai, China). The OSCC cell lines were cultured in DMEM medium containing 10% fetal bovine serum (FBS), 100 U/mL penicillin, 100 U/mL streptomycin and 0.03% glutamine at 37 oC in 5% CO2.

extract_protocol:
After 48 hours, remove the medium and directly add 800μL Trizol into the 6-well plate to harvest samples. Total RNA was extracted using Trizol reagent (Invitrogen, USA) according to the manufacturer’s instructions. The RNA purity and concentration was checked using the NanoPhotometer spectrophotometer (IMPLEN, CA, USA).
A total amount of 1.5 µg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNext Ultra RNA Library Prep Kit for Illumina (NEB, USA) following manufacturer's recommendations and index codes were added to attribute sequences to each sample.

treatment_protocol: The human OSCC cells were exposed to 10 μM DAC for 48 hours. After DAC treatment, cells were harvested for RNA sequencing.

value: OSCC  cell line

value: 10 μM 5-AZA-CdR


title: DMSO_SCC15

description: Library name: Sample 3

value: NC


title: enriched nuclei_MD_5575_7290


title: enriched nuclei_MD_5669_6736


title: enriched nuclei_MD_5669_6739

value: Oligo


title: enriched nuclei_MD_5575_6735


title: enriched nuclei_MD_5575_7980


title: enriched nuclei_MD_5575_6730


title: rats received sacral neural crest cell transplantation following MPG injury, Sacral NC_3

description: Library name: Sacral NC_3


title: enriched nuclei_MD_5575_6728


title: DMSO_CAL27

description: Library name: Sample 1

value: OSCC cell line


title: enriched nuclei_MD_5575_6723

value: Astrocyte


title: DAC_CAL27

description: Library name: Sample 2


title: rats received sacral neural crest cell transplantation following MPG injury, Sacral NC_1

description: Library name: Sacral NC_1


description: Library name: lnjury_3

value: The neurogenic erectile dysfunction model was established by bilaterally clamping the major pelvic ganglia (MPG) in rats, lnjury_3


title: enriched nuclei_MD_5512_7981

value: Cerebellum


title: UTD T cells, Cas9 WT mRNA, 20 gRNAs for 20 distinct genes, 48hrs post EP, donor ND637

city: Philadelphia

first: Regina

middle: M.

last: Young

state: Pennsylvania

address: 3400 Civic Center BLVD

department: CCI

institute: University of Pennsylvania

description: Library name: ND637 UTD Cas9 20x

data_processing:
Sequence reads were trimmed using Trimmomatic v0.36
Reads were aligned to the Homo sapiens GRCh38 reference genome with the STAR aligner v2.5.2b
Unique gene hit counts were obtained using featureCounts from the Subread package v1.5.2, counting only unique reads within exon regions
Assembly: GRCh38
Supplementary files format and content: Unique gene hit counts were calculated by using featureCounts from the Subread package v.1.5.2. The hit counts were summarized and reported using the gene_id feature in the annotation file. Only unique reads that fell within exon regions were counted. If a strand-specific library preparation was performed, the reads were strand-specifically counted.

source_name: primary human T cells

growth_protocol: Primary human T cells were maintained in CTS Optimizer media in humidified atmosphere with 5% CO2 at 37C.

extract_protocol:
Cultured T cells were preserved in RNAprotect Cell Reagent with storage at -80C until processing. Total RNA was extracted using the Qiagen RNeasy Plus Universal Mini Kit.
Strand-specific RNA sequencing libraries were prepared by using NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB)

treatment_protocol: Primary human T cells were activated with Dynabeads CD3/CD28 CTS (Gibco) at a 3:1 bead-to-cell ratio for 48 hrs. Forty-eight hours after stimulation, beads were removed, and T cells were centrifuged at 300g, washed once with PBS, and resuspended at 5 × 10⁶ cells/100 μl in P3 Solution with Supplement (Lonza). 100 µl of cell suspension was combined with 25 µg of Cas9 WT or ABE8e mRNA (8.33 µl of 3 µg/µl solution) and 11.11µl gRNA mix (5 µg per gRNA, up to 100 µg). Electroporation was performed using the Lonza 4D-Nucleofector Core/X Unit and the P3 Primary Cell 4-D Kit (Lonza) using the program EO-115.

value: primary human T cells

value: INDEL formation at 20 distinct sites (CD3E, B2M, CIITA, PVR, CD52, ICAM1, CD58, PDCD1, CTLA4, CD5, RASA2, MED12, ZC3H12A, RC3H1, TET2, ARID1A, SMARCC1, SMARCA4, SMYD3, ADORA2) with gRNAs designed for ABE-mediated KO using WT Cas9

value: 48 hrs after electroporation of editing components


description: Library name: lnjury_2

value: The neurogenic erectile dysfunction model was established by bilaterally clamping the major pelvic ganglia (MPG) in rats, lnjury_2


title: enriched nuclei_MD_5512_7885


title: enriched nuclei_MD_5512_6785


title: UTD T cells, Cas9 WT mRNA, mock, 48hrs post EP, donor ND637

description: Library name: ND637 UTD Cas9 Mock


title: enriched nuclei_MD_5669_6747


description: Library name: lnjury_1

value: The penile tissue of rats

value: The neurogenic erectile dysfunction model was established by bilaterally clamping the major pelvic ganglia (MPG) in rats, lnjury_1


title: rats received sacral neural crest cell transplantation following MPG injury, Sacral NC_2

description: Library name: Sacral NC_2

value: pluripotent stem cells


title: enriched nuclei_MD_5512_6716


title: UTD T cells, ABE8e mRNA, 20 gRNAs for 20 distinct genes, 48hrs post EP, donor ND637

description: Library name: ND637 UTD ABE 20x

value: (functional) KO of 20 distinct genes (CD3E, B2M, CIITA, PVR, CD52, ICAM1, CD58, PDCD1, CTLA4, CD5, RASA2, MED12, ZC3H12A, RC3H1, TET2, ARID1A, SMARCC1, SMARCA4, SMYD3, ADORA2) using adenine base editing


title: Neural stem cells_ shPaupar cells (4)

city: Oxford

first: Farah

last: Alammari

address: Sherrington road

institute: Oxford University

description: Library name: Paupar_4

data_processing:
Original image data file from high-throughput sequencing (like Illumina) is transformed into sequenced reads (called Raw Data or Raw Reads) by CASAVA base recognition (Base Calling). Raw data are stored in FASTQ(fq) format files, which contain sequences of reads and corresponding base quality
Perform alignments with HISAT2 to the reference. HISAT2 uses a graph-based alignment and has succeeded HISAT and TOPHAT2. HISAT2 is a fast and sensitive alignment program for mapping next-generation sequencing reads. Besides one global GFM index, HISAT2 also includes a large set of small GFM indexes that collectively cover the whole genome. These small indexes (local indexes), combined with multiple alignment strategies, enabled effective alignment of RNA-seq reads, particularly, reads spanning multiple exons(Kim D et al., 2015).
Assembly: Stringtie has specific parameters for different libraries, allows accurate assembling of transcripts and quantifies transcripts (Ghosh S et al., 2016) . So we used the Stringtie software to assemble reads into transcripts and quantify them based on comparisons to genomes (Pertea M et al., 2015).
Supplementary files format and content: Excel file includes transcript id, baseMean, log2FoldChange, lfcSE, stat, pvalue, padj

source_name: Brain-SVZ

growth_protocol: Under laminar flow hood, the SVZ tissues were dissected and dissociated with Accutase (Sigma) at 37 °C. Cells were then cultured in Neurobasal medium (Gibco) containing B27 (Gibco), L-glutamine (Invitrogen), Pen Strep (Invitrogen), EGF (20 ng/mL, Sigma), and FGF-2 (20 ng/mL, R&D).

extract_protocol:
Total RNA was isolated using the Qiagen Mini RNeasy kit (Qiagene 74104). DNA was removed using RNase-Free DNase Set (Qiagen 79254).
Total RNA was used as input material for the RNA sample preparations. Sequencing libraries were generated using RNA Library Prep Kit for Illumina following manufacturer’s recommendations and index codes were added to attribute sequences to each sample. Briefly, mRNA was purified from total RNA using probes to remove rRNA. Fragmentation was carried out using divalent cations under elevated temperature in First Strand Synthesis Reaction Buffer(5X). First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNaseH). Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3’ ends of DNA fragments, Adaptor with hairpin loop structure were ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 370~420 bp in length, the library fragments were purified with AMPure XP system (Beverly, USA). Then 3 µL USER Enzyme was used with size-selected, adaptor-ligated cDNA at 37°C for 15 min followed by 5 min at 95 °C before PCR. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, PCR products were purified (AMPure XP system) library quality was assessed on the Agilent 5400 system Agilent USA and quantified by QPCR (1.5 nM). The Qualified libraries were pooled and sequenced on Illumina platforms with PE150 strategy, according to effective library concentration and data amount required.

treatment_protocol: shhRNA Neural stem cell nucleofection was performed following the protocol of LONZA (VPG-1004).

value: Brain-SVZ

value: Neural Stem Cells


title: enriched nuclei_MD_5485_6776


title: UTD T cells, Cas9 WT mRNA, 20 gRNAs for 20 distinct genes, 48hrs post EP, donor ND597

description: Library name: ND597 UTD Cas9 20x


title: enriched nuclei_MD_5512_6784

value: OPC


title: UTD T cells, Cas9 WT mRNA, mock, 48hrs post EP, donor ND597

description: Library name: ND597 UTD Cas9 Mock


title: enriched nuclei_MD_5391_6661


title: Neural stem cells_ shPaupar cells (2)

description: Library name: Paupar_2


title: Neural stem cells_ shPaupar cells (1)

description: Library name: Paupar_1


title: enriched nuclei_MD_5391_6646


title: UTD T cells, ABE8e mRNA, 20 gRNAs for 20 distinct genes, 48hrs post EP, donor ND597

description: Library name: ND597 UTD ABE 20x


title: enriched nuclei_MD_5391_6643


title: enriched nuclei_MD_5391_6674


title: enriched nuclei_MD_5391_6647


title: enriched nuclei_MD_5485_8434


title: enriched nuclei_MD_5485_8319


title: Neural stem cells_ shPaupar cells (3)

description: Library name: Paupar_3


title: enriched nuclei_MD_5391_6639


title: Neural stem cells_ control_cells (4)

description: Library name: Control_4


title: UTD T cells, ABE8e mRNA, mock, 48hrs post EP, donor ND597

description: Library name: ND597 UTD ABE Mock


title: Neural stem cells_ control_cells (3)

description: Library name: Control 3