title: CNCC_ATAC_100_bio3

growth_protocol: Cranial neural crest cells (CNCC) were generated from hESCs as previously described (Prescott et al  2015). In brief, hESC colonies were detached and dissociated using collagenase IV and gentle pipetting. After washing with DPBS, colonies with ~100-200 cells were resuspended in neural crest induction media (1:1 ratio of Neurobasal medium and D-MEM F-12 medium, 0.5 x B27, 0.5 x N2 supplement, 20 ng/ml bFGF, 20 ng/ml EGF, 5 mg/ml bovine insulin and 1 x Glutamax-I supplement and transferred to a 10 cm petri dish (Day 0). By day ~7, neural crest cells started to emerge from the attached neuroectoderm spheres. After an additional 4-5 days, neural crest cells were dissociated with StemPro Accutase and cultured on fibronectin coated 6-well plates in Early Maintenance Media (1:1 Neurobasal medium/D-MEM F-12 medium, 0.5 x B27 supplement, 0.5 x N2 supplement, 20 ng/ml bFGF, 20 ng/ml EGF, 1 x Glutamax-I supplement and 1 mg/ml bovine serum albumin, serum replacement grade. After two passages, Early Maintenance Media was then supplemented with 50 pg/ml BMP2 and 3 mM ChIRON 99021. Cranial neural crest cells were harvested after an additional two to three passages for all downstream experiments.

extract_protocol: ATAC-seq libraries were generated from whole cells as previously described (Calviello et al., 2019). Briefly, cells were dissociated with StemPro Accutase and 100,000 viable cells (as determined using the Muse Cell Analyzer (Merck) ‘Count and Viability’ assay) were washed in ice cold PBS. Cells were pelleted at 500 xg for 5 min at 4 °C and resuspended in transposition reaction mix (25 µL 2 x TD reaction buffer (Illumina), 2.5 µL Tn5 transposase (Illumina) and 22.5 µL sterile water) and incubated at 37 °C in a thermoshaker at 1,400 rpm for 1 hour. The transposition reaction was immediately purified in 10 µL elution buffer using a MinElute reaction clean-up kit (Qiagen). Transposed DNA fragments were then initially amplified using 2.5 µL each of Nextera Index i7 and Nextera Index i5 (Nextera index primer kit, Illumina) with 25 µL NEB Next High Fidelity 2 x PCR master mix (NEB) under the following conditions: 72 ° for 5 min, 98 °C for 30 sec followed by five cycles of 98 °C for 10 sec, 63 °C for 30 sec and 72 °C for 1 min. To reduce GC and size biases, the total PCR cycle was determined as previously described


title: NCC_rep2_day3_ctrl

status: Public on Nov 10 2021

type: SRA

city: Manchester

first: Angela

last: Simeone

address: 200 Hathersage Rd

country: United Kingdom

institute: Genomic Services QIAGEN

library_source: transcriptomic

data_processing:
RNA-seq fastq have been trimmed from adaptors using cutadapt (cutadapt -q 20 -m 20 -a AGATCGGAAGAGC) and alined to hg38 using RSEM (RSEM-1.3.1, rsem-calculate-expression --phred33-quals -p 40 --bowtie2). We used the gene results to further quantify gene expression level in each individual library. TTPM have been generated after quantifying the signal per transcript and scaled by transcript length.
Genes for the differential analysis have been selected only when they showed 1cpm in at least 2 libraries (2 replicates) in any of the experimental conditions. Differential analysis have been performed using EdgeR accounting for the presence of biological replicates. All individual experimental conditions have been contrasted for identifying gened with differential changes.
Differential expressed genes were those with abs(logFC)>2 and FDR<=0.05.
Genome_build: hg38
Supplementary_files_format_and_content: genes.results: count table for gene models based on genecode v.28
Supplementary_files_format_and_content: summary_outcome_all_comparisons.csv: contains the outcome of differential expression. Column names indicate the comparison examined (COND1__VS_COND2. Rows: genes. Cell content: 0 = genes not differentially expressed; 1= genes differentially up-regulaed in COND1; -1: genes differentially down-regulated in COND1.
Supplementary_files_format_and_content: all_cpm.all_libraries.csv: table with all gene counts (expected counts output from Rsem) from all libraries
Supplementary_files_format_and_content: TPM_hg38_phenDC3.csv: table with all gene TPM (Transcript per Million) from all libraries

molecule: total RNA

organism: Homo sapiens

source_name: hESC NCC Differentiation Day3

growth_protocol: Cranial neural crest cells were generated from H1-hESC OCT4-EGFP using a 5 day procedure as previously described (Gomez et al 2019). ROCK inhibitor Y-27632 and ChIRON 99021 was included in the induction media for the first 48 hr only. Induction media was changed daily until day of collection (day 3).

extract_protocol:
Total RNA was extracted from 1 x 10^6 cells using the RNeasy Plus Mini. Kit (Qiagen) as per manufacturer’s instructions. RNA quality (RIN >8) was checked via Bioanalyzer (Aglient) and RNA concentration determined using a Qubit fluorometer (Thermofisher).
Libraries were prepared from 1µg RNA using the NEBNEXT Ultra8482 II Directional RNA Library Prep Kit for Illumina (E7760S) in combination with the NEBNExt Poly(A)mRNA Magnetic isolation module (E7490S) as per manufacturer’s instructions.

tag: cell line of origin

value: H1 ESC OCT4-EGFP

tag: treatment


title: NCC_rep1_day3_dmso

growth_protocol: Cranial neural crest cells were generated from H1-hESC OCT4-EGFP using a 5 day procedure as previously described (Gomez et al 2019). 2µM DMSO was included for the duration of the experiment. ROCK inhibitor Y-27632 and ChIRON 99021 was included in the induction media for the first 48 hr only. Induction media was changed daily until day of collection (day 3).

value: 2 uM DMSO


title: NCC_rep1_day3


title: NCC_rep1_day5_ctrl

source_name: hESC NCC Differentiation Day5

growth_protocol: Cranial neural crest cells were generated from H1-hESC OCT4-EGFP using a 5 day procedure as previously described (Gomez et al 2019). ROCK inhibitor Y-27632 and ChIRON 99021 was included in the induction media for the first 48 hr only. Induction media was changed daily until day of collection (day 5).


title: NSC_Rep3b_and_3_3xBG4_rep3

library_source: genomic

data_processing:
Fastq files were trimmed from adapters using cutadapt (-m 10 -q 20 -O 3 -a CTGTCTCTTATACACATCT) and aligned to a the human genome hg38 with bwa-mem. Bam files were generated from the alignment with samtools view. Duplicated reads were marked and removed using picard MarkDuplicates (ver: 2.20.3).Peak calling was performed with macs2 over input (ver. 2.1.2) with fdr 0.05 (default) and --broad option. Regions confirmed across 2 out of 3 tecnical replicates have been assigned to each biological replicate. Regions shared by 2 out of 3 biological replicates have been elected as consensus of the cell type and multiIntersectBed was used for this. Bigwig files have been generated using deeptools bamCoverage --scaleFactor using the 1000000/total_library_size as a  scaling factor.
ATAC-seq: fastq files were trimmed from adapters with cutadapt ( -a AGATCGGAAGAGC -A AGATCGGAAGAG) and aligned to hg38 with bwa-mem for paired-end (-M -t 12 $read1 $read2). Mapped reads have been filtered using samtools view (-Sb -F780 -q 10 -L )  to keep only mapped and properly paired reds. Those reads have been then used to identify accessible reagions using macs2 wih default options. Regions identified in 2 out 3 biolofical replicates have used as consensus each cell type.
RNA-seq fastq have been trimmed from adaptors using cutadapt (cutadapt -q 20 -m 20 -a AGATCGGAAGAGC) and alined to hg38 using RSEM (RSEM-1.3.1, rsem-calculate-expression --phred33-quals -p 40 --bowtie2). We used the gene results to further quantify gene expression level in each individual library. Technical replicates quantification have been summed up before subsequent analysis. TPM have been generated after quantifying the signal per transcript and scaled by transcript length.
Genome_build: hg38
Supplementary_files_format_and_content: broadPeak: broad peak regions obtained with macs2 ( fdr 0.05 and --broad options)
Supplementary_files_format_and_content: narrowPeak: narrowPeak regions obtained with macs2 default options
Supplementary_files_format_and_content: genes.results.txt: count table for gene models based on genecode v.28
Supplementary_files_format_and_content: *ATAC.multi2.sorted.bed: ATAC consensus regions obtained as those observed in 2 out of 3 biologicial replicates
Supplementary_files_format_and_content: bio2out3.bed: G4 consensus regions obtained as regions observed in 2 out of 3 biological replicates
Supplementary_files_format_and_content: TPM_hg38_all_bio_rep_*.csv: Matrix table with expression levels expressed as TPM (transcript per million) for every gene and every sample. In case of multiple technical replicates TPM have been generated from the data obtained after summing up technical replicates.

molecule: genomic DNA

source_name: neural stem cells (NSC)

growth_protocol: H9-derived neural stem cells (NSC; WiCell) were cultured in StemPro NSC SFM complete media (ThermoFisher) on Geltrex (Thermofisher) pre-coated plates. Media was replenished every two days and NSC were passaged every four to five days using StemPro Accutase (ThermoFisher).

extract_protocol: G4-ChIP-seq was performed as previously described (Hansel-Hertsch et al 2018) with the following modifications. 30 million cells were dissociated with Accutase and cross-linked in serum-free media containing 1% (v/v) methanol-free formaldehyde for 10 min and then quenched with 0.12M glycine for 10 min at room temperature. Cross-linked cells were washed with PBS by centrifugation and flash frozen in liquid nitrogen and stored at -80 °C. Chromatin was isolated using the Lysis and Hypotonic Buffer for Sonication kit (Chromatrap). Each lysed nuclei suspension was sonicated for 25-40 cycles (30 sec on/ 60 sec on, high setting) using a water-cooled bath sonicator until an average DNA size of 100-500 bp was reached.  For ChIP, 7.5 µL of 150 ng/mL chromatin was incubated in Blocking Buffer (25 mM HEPES pH 7.5, 10.5mM NaCl, 110m KCl, 1mM MgCl2 and 1% (w/v) bovine serum albumin) supplemented with 3 µL 1mg/ml RNaseA for 30 min at 800 rpm on a thermoshaker at 16 °C. Next 6 µL of 4.8µM BG4 anti-G4 antibody (generated via autoinduction as previously described in Biffi et al 2014) was added and the reaction incubated for 1 h at 16 °C with mixing at 1,400 rpm. After pre-blocking anti-flag M2 magnetic beads (Merck) with Blocking Buffer for 1 hour at 4°C, 150 µL of this bead slurry solution was then added to the CHIP reaction and incubated for 1 h at 16 °C with mixing at 1,400 rpm. Beads containing antibody bound chromatin were magnetically captured, supernatant discarded via vacuum aspiration and washed four times with wash buffer (10 mM Tris pH 7.4, 100 mM KCl, 0.1% (v/v) Tween 20) at 4°C by manual agitation.  Beads were then subjected to two subsequent washes at 37 °C in a thermoshaker at 1,400 rpm for 10 min. The supernatant was then removed, beads resuspended in 150 µL of elution buffer (1 x TE buffer, 3 µL proteinase K) and incubated at 65 °C  with 1,400 rpm shaking for 3 h to digest protein.  DNA from ChIP and input samples was then purified using MinElute Reaction Clean up Kit. For library preparation, samples were quantified by Qubit dsDNA HS assay and 10ng of purified ChIP or input DNA was tagmentated (2.5 µL Tn5 (Illumina) in tagmentation buffer (Illumina)) in total reaction volume of 40 µL at 800 rpm shaking for 20 min at 37 °C. After purification by MinElute Reaction Clean up Kit, libraries were generated by mixing 20 µL tagmentated DNA, 2.5 µL each of Nextera Index i7 and Nextera Index i5 (Nextera index primer kit, Illumina) with 25 µL NEB Next High Fidelity 2 x PCR master mix (NEB) in a PCR reaction using the following conditions: 72 °C for 5 min, 98 °C for 30 sec followed by six cycles of 98 °C for 10 sec, 63 °C for 30 sec and 72 °C for 1 min. Libraries were purified by MinElute reaction clean-up kit and library quality and quantity were assessed by Bioanalyser and Qubit, respectively. Samples were sequences on a NextSeq platform (single-end, 75 bp reads).

tag: cell type

value: H9 embryonic stem cell-derived neural stem cell

value: H9 ESCs (WA09)

tag: chip antibody

value: BG4 single-chain antibody


title: NSC_Rep3_3xBG4_input


title: ESC_Rep3_3xBG4_input

source_name: human embryonic stem cells (ESC)

value: H9 embryonic stem cells


title: ESC_Rep1_3xBG4_input


title: NCC_rep1_day5_dmso

growth_protocol: Cranial neural crest cells were generated from H1-hESC OCT4-EGFP using a 5 day procedure as previously described (Gomez et al 2019). 2µM DMSO was included for the duration of the experiment. ROCK inhibitor Y-27632 and ChIRON 99021 was included in the induction media for the first 48 hr only. Induction media was changed daily until day of collection (day 5).


title: NSC_Rep1_3xBG4_rep4


title: NSC_Rep1_3xBG4_rep3


title: NSC_Rep2_3xBG4_rep1


title: NSC_Rep1_3xBG4_rep2


title: ESC_Rep3_3xBG4_rep4

growth_protocol: H9 human embryonic stem cells (ESC, WiCell) were cultured in mTESR1 media (StemCell Technologies) and grown on Matrigel matrix (Corning).  ESCs were fed daily and routinely passaged every 4-6 days using ReLeSR (StemCell Technologies).


title: ESC_Rep3_3xBG4_rep2


title: NCC_rep2_day3_phenDC3

growth_protocol: Cranial neural crest cells were generated from H1-hESC OCT4-EGFP using a 5 day procedure as previously described (Gomez et al 2019). 2µM PhenDC3 was included for the duration of the experiment. ROCK inhibitor Y-27632 and ChIRON 99021 was included in the induction media for the first 48 hr only. Induction media was changed daily until day of collection (day 3).

value: 2 uM PhenDC3


title: ESC_Rep3_3xBG4_rep1


title: CNCC_Rep2_3xBG4_input

source_name: cranial neural crest cells (CNCC)

value: H9 embryonic stem cell-derived cranial neural crest cell


title: CNCC_ATAC_100_bio2


title: ESC_Rep1_3xBG4_rep2


title: ESC_Rep2_3xBG4_rep2


title: CNCC_Rep3_3xBG4_rep3


title: NSC_ATAC_50_bio4

extract_protocol: ATAC-seq libraries were generated from whole cells as previously described (Calviello et al., 2019). Briefly, cells were dissociated with StemPro Accutase and 50,000 viable cells (as determined using the Muse Cell Analyzer (Merck) ‘Count and Viability’ assay) were washed in ice cold PBS. Cells were pelleted at 500 xg for 5 min at 4 °C and resuspended in transposition reaction mix (25 µL 2 x TD reaction buffer (Illumina), 2.5 µL Tn5 transposase (Illumina) and 22.5 µL sterile water) and incubated at 37 °C in a thermoshaker at 1,400 rpm for 1 hour. The transposition reaction was immediately purified in 10 µL elution buffer using a MinElute reaction clean-up kit (Qiagen). Transposed DNA fragments were then initially amplified using 2.5 µL each of Nextera Index i7 and Nextera Index i5 (Nextera index primer kit, Illumina) with 25 µL NEB Next High Fidelity 2 x PCR master mix (NEB) under the following conditions: 72 ° for 5 min, 98 °C for 30 sec followed by five cycles of 98 °C for 10 sec, 63 °C for 30 sec and 72 °C for 1 min. To reduce GC and size biases, the total PCR cycle was determined as previously described (Buenrostro et al., 2013). Libraries were purified by AmpureBeads and library quality and quantity were assessed by Bioanalyser and Qubit, respectively. Samples were sequenced on the NextSeq platform (2 x 75 bp).


title: ESC_ATAC_50_bio1

extract_protocol: ATAC-seq libraries were generated from whole cells as previously described (Calviello et al., 2019). Briefly, cells were dissociated with StemPro Accutase and 50,000 viable cells (as determined using the Muse Cell Analyzer (Merck) ‘Count and Viability’ assay) were washed in ice cold PBS. Cells were pelleted at 500 xg for 5 min at 4 °C and resuspended in transposition reaction mix (25 µL 2 x TD reaction buffer (Illumina), 2.5 µL Tn5 transposase (Illumina) and 22.5 µL sterile water) and incubated at 37 °C in a thermoshaker at 1,400 rpm for 1 hour. The transposition reaction was immediately purified in 10 µL elution buffer using a MinElute reaction clean-up kit (Qiagen). Transposed DNA fragments were then initially amplified using 2.5 µL each of Nextera Index i7 and Nextera Index i5 (Nextera index primer kit, Illumina) with 25 µL NEB Next High Fidelity 2 x PCR master mix (NEB) under the following conditions: 72 ° for 5 min, 98 °C for 30 sec followed by five cycles of 98 °C for 10 sec, 63 °C for 30 sec and 72 °C for 1 min. To reduce GC and size biases, the total PCR cycle was determined as previously described


title: NSC_Rep3_3xBG4_rep4


title: CNCC_Rep1_3xBG4_input


title: NSC_Rep3_3xBG4_rep3


title: NSC_Rep3_3xBG4_rep2


title: CNCC_Rep2_3xBG4_rep1


title: C1011

city: Cambridge

first: Sergio

last: Martínez Cuesta

address: 1 Francis Crick Avenue

department: Data Sciences and Quantitative Biology, Discovery Sciences

institute: AstraZeneca

description:
Polysome profiling - 10uM PDS treated
polysome_htseq_gene_counts.txt

data_processing:
Library strategy: Polysome profiling
The data was processed as described in the methods section of the associated manuscript. Details about the analysis are available in https://github.com/sblab-bioinformatics/rna-g4-proteins
Genome_build: GRCh38, hg19
Supplementary_files_format_and_content: The DHX36 and GRSF1 iCLIP-seq data were processed jointly with the WT DDX3X iCLIP-seq data available in GSE106476 (GSM2838585, GSM2838586, GSM2838587).

growth_protocol: Flp-In T-REx 293 (ThermoFisher) and HeLa (ATCC) cells were cultured in Dulbecco’s modified Eagle’s medium containing L-Glutamine (Sigma) supplemented with 10% fetal bovine serum (FBS; Gibco) or Tetracycline-free FBS (Clonetech) at 37oC and 5% CO2. Inducible stable cell lines expressing Strep-tag (ST)/HA affinity tagged GRSF1 and DHX36 were generated by transfection of pcDNA5/FRT/TO vector as per manufacturer's protocol (ThermoFisher). Cells were selected with Blasticidin (Gibco) and Hygromycin D (Sigma).

extract_protocol:
iCLIPs were performed as described in Herdy et al., Nucleic acids Res., 2018. For cRIP, cells were UV crosslinked and cytoplasmic lysates collected by hypotonic lysis. RNA was fragmented with RNaseA and subjected to affinity enrichment using BG4 or A9 antibody. RNA fragments were purified following de-crosslinking and proteinase K digestion using phenol/chloroform. Polysome profiling was performed as described in Herdy et al., Nat. Immunology, 2012. RNA was isolated using Trizol reagent (LifeTechnologies).
iCLIP libraries were prepared as described in Huppertz et al., Methods, 2014. cRIP libraries were prepared using NEBNext® rRNA depletion kit and NEBNext® Ultra™ II Directional RNA Library Prep Kit. Polysome profiling libraries were prepared using TruSeq RNA library prep kit (Illumina).

treatment_protocol: For polysome profiling, cells were treated for 45mins with PDS or DMSO.

value: HeLa

tag: genotype

value: WT

value: 10uM PDS

tag: genome build

value: GRCh37 (hg19)


title: NSC_Rep2_3xBG4_rep4


title: NSC_Rep2_3xBG4_rep2


title: ESC_Rep2_3xBG4_rep4


title: ESC_Rep2_3xBG4_rep3


title: ESC_Rep1_3xBG4_rep4


title: ESC_ATAC_50_bio4


title: B2L

description:
Polysome profiling - 2uM PDS treated
polysome_htseq_gene_counts.txt

value: 2uM PDS


title: B211


title: B10L


title: ESC_Rep1_3xBG4_rep1


title: BML

description:
Polysome profiling - DMSO treated
polysome_htseq_gene_counts.txt

source_name: BML

value: DMSO


title: A1011


title: CNCC_Rep3_3xBG4_rep4


title: CNCC_ATAC_100_bio1


title: CNCC_Rep3_3xBG4_rep2


title: CNCC_Rep1_3xBG4_rep4


title: CNCC_Rep2_3xBG4_rep4


title: CNCC_Rep2_3xBG4_rep3


title: AML

source_name: AML


title: CNCC_Rep1_3xBG4_rep2


title: C10L


title: C211


title: C2L


title: CNCC_Rep1_3xBG4_rep1


title: CM11


title: CML

source_name: CML


title: DHX36_WT2

data_processing:
Library strategy: iCLIP-seq
The data was processed as described in the methods section of the associated manuscript. Details about the analysis are available in https://github.com/sblab-bioinformatics/rna-g4-proteins
Genome_build: GRCh38, hg19
Supplementary_files_format_and_content: The DHX36 and GRSF1 iCLIP-seq data were processed jointly with the WT DDX3X iCLIP-seq data available in GSE106476 (GSM2838585, GSM2838586, GSM2838587).

value: Flp-In T-REx 293


title: A211


title: A10L


title: AM11


title: DHX36_WT3


title: INPUT_3

description:
RIP-seq input
BG4.peaks.bed

data_processing:
The data was processed as described in the methods section of the associated manuscript. Details about the analysis are available in https://github.com/sblab-bioinformatics/rna-g4-proteins
Genome_build: GRCh38, hg19
Supplementary_files_format_and_content: The DHX36 and GRSF1 iCLIP-seq data were processed jointly with the WT DDX3X iCLIP-seq data available in GSE106476 (GSM2838585, GSM2838586, GSM2838587).

tag: antibody


title: BM11


title: GRSF1_WT4


title: GRSF1_WT1


title: B1011


title: DHX36_WT1


title: INPUT_2

description:
RIP-seq input
BG4.peaks.bed


title: BG4_2


title: A9_3


title: A2L


title: A9_2


title: GRSF1_WT2


title: HD5

city: Lausanne

first: Marjan

last: Biočanin

state: Vaud

address: Station 19, SV 3820

department: SV-IBI

country: Switzerland

institute: EPFL

data_processing:
Drop-seq tools 2.3.0 package on the EPFL SCITAS HPC platform. After trimming and sequence tagging, reads were aligned to the mouse reference genome (mm10) using STAR (version 2.7.0.e). https://github.com/mccrowjp/Dropseq
Following the alignment, the gene annotation was added, bead synthesis errors were corrected and cell barcodes extracted. Subsequently, the BAM files containing the processed data were used to obtain digital gene expression matrices.
Downstream data analysis was carried out using R (version 3.5.0)
Genome_build: hs.g38r91
Supplementary_files_format_and_content: tab-delimited text files include read-count matrices for each experiment and digital gene expression files

source_name: HEK 293T

growth_protocol: The human embryonic kindley 293T cell line was cultured to 90% confluency in Glutamax DMEM supplemented with FBS and penicillin-streptomycin.

extract_protocol:
Prior to use, cells were washed with PBS, dissociated via Trypsin-EDTA, washed with PBS, BSA and counted with Trypan blue live-dead stain using Countess cell counter (Invitrogen). Cells were mixed in a 1:1 ratio, adjusted to 20 cells/µl, and re-suspended in a cell buffer containing PBS, BSA, and Optiprep previous to loading onto the DisCo chip.
After bead-cell in droplet co-encapsulation, the gel loading tip containing the sample droplets was transferred to a bead collection chip inlet (cp-chip; as described in Lab on a chip). Droplets in the tip were flushed to bead collection chip. Subsequently to bead capture, washing was performed as in Drop-seq with SSC and Reverse transcription buffer directly in the recovery chip. Reverse transcription solution was added to the beads and the recovery chip was placed on a heating block to perform first strand cDNA synthesis (RT) for 90 minutes at 42oC. After RT reaction beads were washed on the recovery chip with TE-SDS once, with TE-TW twice and with Tris once. The beads were treated with Exonuclease I for 45 minutes at 37oC to remove single-stranded oligonucleotides on beads. After Exonuclease I treatment, beads were washed with TE-SDS once, with TE-TW twice (as after reverse transcription). Beads were then eluted from the recovery chip in dH2O. cDNA was amplified for 18 – 23 cycles using Kapa HiFi Hot start ready mix. cDNA was purified with CleanPCR magnetic beads using 0.6X ratio to remove small cDNA fragments and primers. To assess the cDNA yield and quality concentration was measured using Qubit and cDNA traces quality was assessed using Fragment Analyzer (Agilent). cDNA was tagmented with in-house Tn5 (generated as described in Picelli et al..) and the library was purified using CleanPCR magnetic beads (0.6X ratio).
Libraries were sequenced on a NextSeq 500 system (Illumina) following recommendations from original protocol (20 bp for read 1 and 50 bp for read2).

tag: culture method

value: In vitro cell culture

tag: passages

tag: cell line

value: HEK 293T


title: A9_1


title: BG4_3


title: HD2


title: HD1


title: BG4_1


title: HD4


title: GRSF1_WT3


title: HD3


title: INPUT_1

description:
RIP-seq input
BG4.peaks.bed


title: all_ULA-MK-s0010

first: Ludovic

last: Telley

address: Department of Biomedical Sciences University of Lausanne Rue du Bugnon 9

institute: UNIL

description: replicate 2

data_processing:
Reads are mapped and gene expression are generated with Salmon Alevin using the mouse genome GRCm38 considering M25 genecode release.
The deduplicate of the reads with identical cell barcode sequence was done by providing to Salmon Alevin a whitelist of barcodes (whitelist.txt)
Command line use for Alevin : salmon alevin -l ISR -i txintron/GRCm38.primary_assembly.transcripts.introns.sidx -1 $(word 1,$^) -2 $(word 2,$^) -o $@ -p 8 --umiLength 6 --barcodeLength 8 --end 3 --dumpFeatures --tgMap txintron/GRCm38.primary_assembly.transcripts.introns.tx2gene.tsv --whitelist whitelist.txt
Genome_build: GRCm38
Supplementary_files_format_and_content: singlecell_countmatrix.tsv.gz: is a count matrix in tab-separated format. It contains the result of the single cell quantification for all genes. The  values represent the number of exonic reads mapping into each gene after UMI correction. The first columns contain the gene name that is quantified.    singlecell_metadata.tsv: is a table containing metadata information for each 304 cells analyse in the paper in tab-separated.    whitelist.txt : is a text file containing barcode id information to deduplicated fastq files.
Supplementary_files_format_and_content: singlecell_countmatrix.tsv.gz: raw counts
Supplementary_files_format_and_content: singlecell_metadata.tsv: tab-delimited text file includes meta information for each single cell
Supplementary_files_format_and_content: whitelist.tsv: text file includes cell barcode ID for demultiplexing fastq

molecule: polyA RNA

organism: Mus musculus

source_name: Neural stem/progenitor cells (NSPCs)

growth_protocol: Adult mouse NSPCs from the SVZ of 6-8-week-old C57/Bl6 male mice were isolated using the papain-based MACS Neural Tissue Dissociation Kit (Milteny, #130-092-628), according to the manufacturer’s instructions. The resulting single cell suspensions were cultured as neurospheres in DMEM/F12/GlutaMAX (Invitrogen, #31331-028) complemented with B27 (Invitrogen, #17504044), 20 ng/ml human EGF (AF-100-15, PeproTech), 20 ng/ml human basic FGF-2 (100-18B, PeproTech), and 1x PSF (Invitrogen, #15240062). Medium was changed every 2-3 days. The neurospheres were split a minimum of 5 passages to remove progenitors or other proliferating cells. The cells were then adapted to the same medium supplemented with N2 instead of B27, cultured on on uncoated standard plastic cell culture dishes (Corning, #430167, 100mm*20mm, TC-treated) and medium was changed every 2-3 days. The cells were stably transfected with a PLIN2-GFP retrovirus and kept as a stable line for several passages prior to the scRNA Seq experiment

extract_protocol:
Proliferating SVZ PLIN2-GFP NSPCs were trypsinized and incubated for 10min at 37°C with the cell viability dye Calcein Red (C34851 ThermoFisher, 1:1000 in 1x PBS). Cells were washed twice with PBS and kept in EDTA-DPBS (E8008, Merck Millipore) at 4°C during the sorting process (Sort-seq). SORT-seq: Single-cell mRNA sequencing was performed at Single Cell Discoveries (single cell sequencing service, the Netherlands) according to an adapted version of the SORT-seq protocol, with previously described primers. Equal numbers of NSPCs with the 25% highest and lowest GFP signal were collected into 384-well plates containing 384 primers and mineral oil (Sigma). After sorting, plates were snap-frozen on dry ice and stored at -80° C.
For amplification cells were heat-lysed at 65° C followed by cDNA synthesis using steps based on CEL-Seq2 protocol After second strand cDNA synthesis, barcoded material was pooled into libraries of 384 cells and amplified using IVT. Following amplification, library preparation was processed according to CEL-seq2 protocol where libraries were finally multiplexed using TruSeq small RNA primers (Illumina). Paired-end sequencing was achieved on Illumina Nextseq 500 (Read 1: 26 cycles, index read: 6 cycles, Read 2: 60 cycles).

value: Neural stem/progenitor cells


title: S6_0.2

data_processing:
Drop-seq tools package on the EPFL SCITAS HPC platform. After trimming and sequence tagging, reads were aligned to the mouse  reference genome (mm10) using STAR (version 2.7.0.e). https://github.com/mccrowjp/Dropseq
Following the alignment, the gene annotation was added, bead synthesis errors were corrected and cell barcodes extracted. Subsequently, the BAM files containing the processed data were used to obtain digital gene expression matrices.
Downstream data analysis was carried out usingR (version 3.5.0) using Seurat (version 3.1.1) and uwot (version 0.1.3).
Genome_build: mm10
Supplementary_files_format_and_content: tab-delimited text files include read-count matrices for each organoid

source_name: Intestinal organoid

growth_protocol: Intestinal organoids used were isolated from mouse crypts from dissociated mouse intestinal stem cell colonies from 5-10 week old heterozygous Lgr5-eGFP-IRES-CreERT2 mice. Isolation of Lgr5 eGFP+ stem cell population and initial culture was performed as described elsewhere. For developmental experiments, organoids were dissociated into cell suspensions and cells were embedded in Matrigel.

extract_protocol:
After bead-cell in droplet co-encapsulation, the gel loading tip containing the sample droplets was transferred to a bead collection chip inlet (cp-chip; as described to in Lab on a chip). Droplets in the tip were flushed to bead collection chip. After beads were captured, washing was performed as in Drop-seq with 6X SSC and Reverse transcription buffer using the same tip. Reverse transcription solution was added to beads and recovery chip was placed on a heating block.
After bead-cell in droplet co-encapsulation, the gel loading tip containing the sample droplets was transferred to a bead collection chip inlet (cp-chip; as described in Lab on a chip). Droplets in the tip were flushed to bead collection chip. Subsequently to bead capture, washing was performed as in Drop-seq with SSC and Reverse transcription buffer directly in the recovery chip. Reverse transcription solution was added to the beads and the recovery chip was placed on a heating block to perform first strand cDNA synthesis (RT) for 90 minutes at 42oC. After RT reaction beads were washed on the recovery chip with TE-SDS once, with TE-TW twice and with Tris once. The beads were treated with Exonuclease I for 45 minutes at 37oC to remove single-stranded oligonucleotides on beads. After Exonuclease I treatment, beads were washed with TE-SDS once, with TE-TW twice (as after reverse transcription). Beads were then eluted from the recovery chip in dH2O. cDNA was amplified for 18 – 23 cycles using Kapa HiFi Hot start ready mix. cDNA was purified with CleanPCR magnetic beads using 0.6X ratio to remove small cDNA fragments and primers. To assess the cDNA yield and quality concentration was measured using Qubit and cDNA traces quality was assessed using Fragment Analyzer (Agilent). cDNA was tagmented with in-house Tn5 (generated as described in Picelli et al..) and the library was purified using CleanPCR magnetic beads (0.6X ratio).
Libraries were sequenced on a NextSeq 500 system (Illumina) following recommendations from original protocol (20 bp for read 1 and 50 bp for read2).

tag: sample type

value: Ex vivo cell culture

tag: batch

tag: tissue

value: organoid


title: all_ULA-MK-s009

description: replicate 1


title: S3_03


title: S5_02


title: S6_01


title: S3_01


title: S3_02


title: S2_02b


title: Crypte19

source_name: Intestinal crypts

growth_protocol: Intestinal crypts used were isolated from mouse small intestine. Single crypts were hand-picked and dissociated to cell suspensions. Cell suspensions for bulk samples were obtained from a random sample of non-handpicked crypts.

value: Ex vivo

value: single crypt


title: Cryptd17


title: S2_01


title: Crypte21


title: Crypte20


title: Cryptd16


title: Cryptc12


title: Cryptc9


title: Cryptc10


title: Cryptc11


title: S5_01


title: Cryptb7


title: S2_02a


title: Cryptb6


title: Cryptd15


title: Cryptb4


title: Crypte18


title: Crypta3


title: Crypta2


title: Bulke4

value: several crypts


title: Crypta1


title: Bulkd3


title: Bulkc2


title: Cryptd13


title: Bulkb1


title: Cryptd14


title: Bulka1


title: Cerebral Cortex Female 5xFAD; Abi3 knockout - rep 5

city: Indianapolis

first: Jungsu

last: Kim

state: IN

address: 320 W. 15th Street

country: USA

institute: Indiana University School of Medicine

data_processing:
Base calling done by Illumina Real-Time Analysis v2
Adapters and polyA tails removed by bbduk from bbtools/38.72 using references files polyA/polyT (poly(A) sequence of 18 As, and a poly(T) sequence of 18 Ts) & truseq_rna.fa (from the BBTools suite: https://sourceforge.net/projects/bbmap/) and parameters k=13 ktrim=r useshortkmers=t mink=5 qtrim=r trimq=10 minlength=20
Alignment done by STAR/2.7.3a using parameters: --outFilterType BySJout --outFilterMultimapNmax 20 --alignSJoverhangMin 8 --alignSJDBoverhangMin 1 --outFilterMismatchNmax 999 --outFilterMismatchNoverLmax 0.6 --alignIntronMin 20 --alignIntronMax 1000000 --alignMatesGapMax 1000000 --outSAMattributes NH HI NM MD
Gene counts done by htseq-count from htseq/0.9.1-py13 using parameters htseq-count -m intersection-nonempty -s yes -f bam -r pos
Genome_build: GRCm39
Supplementary_files_format_and_content: Matrix table of raw gene counts for each gene and each sample in experimental design

source_name: Cerebral cortex

extract_protocol:
Total RNA was extracted from cortical tissues using TRI Reagent® (MRC). Briefly, mouse cortices were homogenized in TRI Reagent using a hand grinder. After 5 min incubation, chloroform was added onto the samples. Samples were centrifuged at 17,000g for 15 min at 4C. Top layer was transferred onto a new tube, processed with isopropanol for RNA precipitation. Samples were centrifuged and RNA pellets were washed with ethanol. RNA pellets were dissolved in nuclease free water and RNA concentration was determined using the Nanodrop 2000 Spectrophotometer (Thermo Fisher).
Isolated RNA was shipped to Lexogen for library preparation and sequencing. Libraries were prepared manually according to the manufacturer’s instructions using QuantSeq 3' mRNA-Seq FWD Library Prep Kit (Lexogen, UG Version 015UG009V0252). Briefly, 50 ng total RNA was denatured for 3 minutes at 85°C with oligo(dT) primers containing an Illumina-compatible sequence at its 5’ end followed by reverse transcription at 42°C for 15 minutes. After RNA removal, the second strand of the cDNA was synthesized by a random primer containing an Illumina-compatible linker sequence at its 5’ end, and the end product was purified by a magnetic bead-based method to remove all reaction components. Finally, individual sample barcodes (dual indexing) for multiplexing were introduced via PCR (cycle number determined by qPCR) and the leftover reaction components were removed by a magnetic bead-based purification method. All libraries were analyzed for adapter dimers, size distribution and concentration on a Fragment Analyzer System using the DNF-474 HS NGS Fragment kit (1-6,000 bp) (Agilent). After pooling the libraries in an equimolar ratio, the concentration and the size distribution of the lane mix was analyzed by Qubit dsDNA HS assay (Thermo Fisher Scientific) and by 2100 Bioanalyzer device using the HS-DNA assay (Agilent), respectively. A 2 nM dilution of the lane mix was denatured and diluted to loading concentration for sequencing on a NextSeq 500 instrument with SR75 High Output Kit (Illumina). Seventy-six-base-pair single-end reads were generated at Lexogen Services.

tag: strain

value: cerebral cortex

tag: age

value: 8 months

value: 5xFAD; Abi3 KO

tag: Sex

value: Female


title: Cerebral Cortex Female 5xFAD; Abi3 knockout - rep 2


title: Cerebral Cortex Female 5xFAD; Abi3 knockout - rep 3


title: Cerebral Cortex Female 5xFAD; Abi3 knockout - rep 4


title: Cerebral Cortex Female 5xFAD; Abi3 knockout - rep 1


title: Cryptb8


title: Cryptb5


title: Cerebral Cortex Female 5xFAD; Abi3 wildtype - rep 4

value: 5xFAD; Abi3 WT


title: Cerebral Cortex Female 5xFAD; Abi3 wildtype - rep 5


title: Cerebral Cortex Female 5xFAD; Abi3 wildtype - rep 1


title: Cerebral Cortex Female 5xFAD; Abi3 wildtype - rep 2


title: Cerebral Cortex Female 5xFAD; Abi3 wildtype - rep 6


title: KO-Mac_biorep_1_lib_TW915

city: SAINT LOUIS

first: Daofeng

last: Li

state: MO

address: 4515 McKinley Avenue

department: Genetics

institute: Washington University in St. Louis

data_processing:
raw reads were processed using the juicer (Cell Syst. 2016 Jul; 3(1): 95–98.) pipline (v1.6), with mm10 as genome and MboI as cut site.
Contact matrices were visualized using WashU epigenome browser, juicebox, and UCSC genome browser, with VC_SQRT as normalization method.
V4C tracks were generated using a custom R script based on the methods detailed in Choudhary et al. 2020. Genome Biology (doi:10.1186/s13059-019-1916-8)
all replicates of the same sample type were aggregared using juicer's mega.sh
Genome_build: mm10
Supplementary_files_format_and_content: inter.hic files: contact matrices from juicer pipeline; inter.txt files: sequencing quality metrics from juicer pipeline

source_name: spleen

extract_protocol:
As detailed in In situ Hi-C: Protocol and Quality Control Standards, 4D Nucleome Consortium (https://drive.google.com/file/d/1wEAR_yGKMFBZ6BQ6CRRtBa8USYdRZahM/view), and in Choudhary et al. 2020. Genome Biology (doi:10.1186/s13059-019-1916-8)
library strategy: Hi-C2

value: Macrophage

value: spleen


title: KO-Mac_biorep_2_lib_TW925


title: KO-Mac_biorep_1_lib_TW916


title: Cerebral Cortex Female 5xFAD; Abi3 wildtype - rep 3


title: WT-Mac_biorep_2_lib_TW924

value: WT


title: KO-Mac_biorep_2_lib_TW926


title: WT-Mac_biorep_2_lib_TW923


title: WT-Mac_biorep_1_lib_TW918


title: pDC-WT_biorep_3_lib_TW922

value: pDC


title: pDC-WT_biorep_3_lib_TW921


title: WT-Mac_biorep_1_lib_TW917


title: pDC-WT_biorep_2_lib_TW920


title: pDC-WT_biorep_1_lib_TW914


title: pDC-WT_biorep_1_lib_TW913


title: GTAC_WT_CD8_TruSeq_1_ATCACGATCT

city: St. Louis

first: Xiao

last: Huang

state: Missouri

address: 4940 Parkview Place, CSRB 7766, Department of Pathology and Immunology

department: Pathology and Immunology

institute: Washington University in St. Louis, School of Medicine

data_processing:
All the Hi-C data was processed using the computational pipeline described before(Rao et al., 2014)
Genome_build: mm10
Supplementary_files_format_and_content: All Hi-C contact map were normalized using VC_SQRT method.
Supplementary_files_format_and_content: hic files

source_name: Splenic CD8 T cells

growth_protocol: Primary cells was sorted from spleen

extract_protocol:
Freshly sorted cells were kept on ice and typically diluted to 100K-1M cells/ml in media (min volume of 1ml). The cells were immediately crosslinked with 2% formaldehyde for 10 min, quenching with 0.2M glycine for 5 min at room temperature and 5 min on ice, followed by permeabilizing them with nuclei intact, digesting the DNA with MboI (4-cutter restriction enzyme), filling the 5′-overhangs while incorporating biotin-14-dATP (a biotinylated nucleotide), followed by ligating the resulting blunt-end fragments, shearing the DNA to a 400–700-bp fragment size using a Covaris ME220 focused ultra-sonicator, capturing the biotinylated ligation junctions with streptavidin beads.
Libraries were prepared according to Illumina's instructions with 10–12 rounds of PCR amplification, and finally analyzed the resulting fragments with paired-end sequencing.

value: FACS sorted splenic CD8+ T cells

value: untreated


title: GTAC_KO_CD8_TruSeq_3_TTAGGCATCT


title: pDC-WT_biorep_2_lib_TW919


title: WT-S Rep2

city: Durham

first: Perry

last: Blackshear

state: NC

address: 111 TW Alexander Drive

department: STL

institute: NIH/NIEHS

data_processing:
Sequenced reads were filtered for low-quality sequence, then mapped to mm10 whole genome using STAR (v2.7.0f).
The featureCounts per gene were calculated using the featureCounts command available from Subread (v1.6.4).
Differential expression analysis was peformed with R package DESeq2.
Genome_build: mm10
Supplementary_files_format_and_content: tab-delimited text file includes featureCounts values for each Sample.

source_name: WT-S

extract_protocol:
RNA was isolated from a 4mm biopsy of the gastric corpus lesser curvature. RNA was extracted in TRIzol (Thermo Fisher Scientific).
RNA libraries were prepared for sequencing using Illumina Tru-Seq RNA Library Preparation Kit

value: 8 weeks

value: spasmolytic polypeptide-expressing metaplasia (SPEM)

value: Stomach


title: WT-S Rep4


title: WT-A Rep4

source_name: WT-A

value: adrenalectomy (ADX)-induced gastric inflammation


title: WT-S Rep1


title: WT-A Rep2


title: TTP-S Rep4

source_name: TTP-S

value: congenic C57BL/6

value: TTP{delta}ARE


title: TTP-S Rep1


title: TTP-S Rep3


title: TTP-A Rep3

source_name: TTP-A


title: TTP-S Rep2


title: TTP-A Rep2


title: N_7191_het_p

city: Dresden

first: Francis

last: Stewart

address: Tatzberg 47-49

country: Germany

institute: BIOTEChnology Center TU Dresden

data_processing:
Basecalling: bcl2fastq2 v. 2.19.1
Read alignment: GSNAP version 2019-06-10 called with args: gsnap.sse42 -D /group/dcgc/sequencing_processed/support/pipeline/gmap -d mm10 --gunzip -A sam -t 12 --use-sarray=1 --input-buffer-size=500000 --output-buffer-size=500000 -B 5 -n 1 -m 0.6 -s EnsemblGene-92.ss.mm10.iit -N 0 --read-group-id=L41990_N_7191_het_p --read-group-name=N_7191_het_p --read-group-library=L41990 --read-group-platform=illumina --action-if-cigar-error noprint fastq/L41990_N_7191_het_p_R1.fastq.gz
featureCounts v2.0.1; Command:"featureCounts" "-a" "/group/dcgc/sequencing_processed/support/pipeline/annotation/mus_musculus/mm10/EnsemblGene-92.mm10.TR.gtf" "-s" "0" "-o" "genecount/bfx1376.mm10.e92.txt" "-Q" "1" "-T" "8" "--tmpDir" "/tmp/856312.1.all.q" "gsnap/L37131_A473_het_SC.bam" "gsnap/L37132_A474_hom_SC.bam" "gsnap/L37133_A475_het_SC.bam" "gsnap/L37134_A476_hom_SC.bam" "gsnap/L37135_A469_hom_SC.bam" "gsnap/L37136_A470_het_SC.bam" "gsnap/L37137_A471_het_SC.bam" "gsnap/L37138_A472_hom_SC.bam" "gsnap/L38855_A473_het_P.bam" "gsnap/L38856_A474_hom_P.bam" "gsnap/L38857_A475_het_P.bam" "gsnap/L38858_A476_hom_P.bam" "gsnap/L38859_A469_hom_P.bam"    "gsnap/L38860_A470_het_P.bam" "gsnap/L38861_A471_het_P.bam" "gsnap/L38862_A472_hom_P.bam" "gsnap/L41978_N_7068_het_sc.bam" "gsnap/L41979_N_7069_hom_sc.bam" "gsnap/L41980_N_7121_hom_sc.bam" "gsnap/L41982_N_7124_het_sc.bam" "gsnap/L41985_N_7182_het_sc.bam" "gsnap/L41986_N_7191_het_sc.bam" "gsnap/L41987_N_7193_hom_sc.bam" "gsnap/L41981_N_7121_hom_p.bam" "gsnap/L41983_N_7124_het_p.bam" "gsnap/L41988_N_7179_hom_p.bam" "gsnap/L41989_N_7182_het_p.bam" "gsnap/L41990_N_7191_het_p.bam" "gsnap/L41991_N_7193_hom_p.bam"
The raw read counts (absolute number of reads) were normalized with the DESeq2 R package (v. 1.22.2)(Anders and Huber, 2010)
Genome_build: mm10
Supplementary_files_format_and_content: bfx1376.mm10.e92.txt.original.csv: tab delimited file with raw readcounts per gene (rows) and sample (columns) (featureCounts output)

source_name: small intestine

extract_protocol:
At 4 and 10 days after the induction of mutagenesis, intestinal crypts were dissociated into single cells with TrypLE Express (Thermo Fisher Scientific) for 30min at 37°C. Dissociated cells were passed through 70µm cell strainer and washed wit 5% FBS/PBS. Cells were stained with PE-conjugated anti-CD24,  APC-conjugated anti-CD326 (EpCAM) antibody and Alexa-Fluor 700 CD45 antibodies for 45min on ice. Stained cells were collected by centrifugation and resuspended in SYTOX blue dead cell stain. FACS sorting was performed on a FACS AriaTM III cell sorter (BD).
Cells are FACsorted into a PCR tube containing 2 ul of nuclease free water with 0.2% Triton-X 100 and 4 U murine RNase Inhibitor (NEB), spun down and frozen at -80°C. After thawing the samples, RNA is denatured for 3 minutes at 72 °C in the presence of 2.4 mM dNTP (Invitrogen), 240 nM dT-primer* and 4 U RNase Inhibitor (NEB). The reverse transcription and addition of the template switch oligo is performed at 42°C for 90 min after filling up to 10 µl with RT buffer mix for a final concentration of 1x superscript II buffer (Invitrogen), 1 M betaine, 5 mM DTT, 6 mM MgCl2, 1 µM TSO-primer*, 9 U RNase Inhibitor and 90 U Superscript II. The reverse transcriptase is inactivated at 70 °C for 15 min. The single stranded cDNA is subsequently amplified using Kapa HiFi HotStart Readymix (Roche) at a 1x concentration together with 250 nM UP-primer* under following cycling conditions: initial denaturation at 98°C for 3 min, 16 cycles [98°C 20 sec, 67°C 15 sec, 72°C 6 min] and final elongation at 72°C for 5 min. The amplified cDNA is purified using 1x volume of Sera-Mag SpeedBeads (GE Healthcare) resuspended in a buffer consisting of 10 mM Tris, 20 mM EDTA, 18.5 % (w/v) PEG 8000 and 2 M sodium chloride solution. The cDNA quality and concentration is determined with the Fragment Analyzer (Agilent). For library preparation 1.5 ng cDNA was tagmented using 0.5 µl TruePrep Tagment Enzyme V50 and 1x TruePrep Tagment Buffer L (TruePrep DNA Library Prep Kit V2 for Illumina, Vazyme), followed by an incubation step at 55 °C for 10 min. Next, Illumina indices are added during PCR (72 °C 3 min, 98 °C 30 sec, 12 cycles [98 °C 10 sec, 63 °C 20 sec, 72 °C 1 min], 72°C 5 min) with 1x concentrated KAPA Hifi HotStart Ready Mix and 300 nM dual indexing primers. After PCR, libraries are purified twice with 1x volume Sera-Mag SpeedBeads and quantified with the Fragment Analyzer, followed by Illumina sequencing on a Nextseq500 with a sample sequencing depth of 30 mio reads on average.

treatment_protocol: Mutagenesis was induced in mice by intraperitoneal injections of 1mg  tamoxifen on 3 consecutive days.

tag: time point

value: 10 days

tag: genotype/variation

value: Paneth cell


title: TTP-A Rep1

tag: treatment


title: TTP-A Rep4


title: N_7193_hom_p

tag: cell type


title: N_7182_het_p


title: N_7193_hom_sc

value: stem cell


title: N_7179_hom_p


title: N_7124_het_p


title: N_7191_het_sc


title: N_7124_het_sc


title: N_7121_hom_sc


title: N_7121_hom_p


title: WT-S Rep3

value: Female


title: Bulka2

value: Ex vivo

value: several crypts


title: A472_hom_P

value: 4 days


title: N_7069_hom_sc


title: A470_het_P


title: WT-A Rep3

value: WT


title: A471_het_P


title: A469_hom_P


title: WT-A Rep1


title: A472_hom_SC


title: A470_het_SC


title: A476_hom_P


title: A469_hom_SC


title: A475_het_P


title: N_7182_het_sc


title: A473_het_P


title: A476_hom_SC


title: A474_hom_SC


title: A475_het_SC


title: A473_het_SC


title: A474_hom_P


title: S3h

data_processing:
Drop-seq tools 2.3.0 package on the EPFL SCITAS HPC platform. After trimming and sequence tagging, reads were aligned to the mouse reference genome (mm10) using STAR (version 2.7.0.e). https://github.com/mccrowjp/Dropseq
Following the alignment, the gene annotation was added, bead synthesis errors were corrected and cell barcodes extracted. Subsequently, the BAM files containing the processed data were used to obtain digital gene expression matrices.
Downstream data analysis was carried out usingR (version 3.5.0) using Seurat (version 3.1.1) and uwot (version 0.1.3).
Genome_build: mm10
Supplementary_files_format_and_content: tab-delimited text files include read-count matrices for each organoid, Seurat-Object

growth_protocol: Intestinal organoids used were isolated from mouse crypts from dissociated mouse intestinal stem cell colonies from 5-10 week old heterozygous Lgr5-eGFP-IRES-CreERT2 mice. Isolation of Lgr5 eGFP+ stem cell population and initial culture was performed as described elsewhere. For the developmental time-course experiments, organoids were dissociated to single-cells, live Lgr5+-eGFP cells isolated using a FACS ARIA II (BD) and embedded in Matrigel. After matrigel polymerization, cells were cultured in DMEM/F12 supplemented with Glutamine, Hepes, penicillin-streptomycin, B27 supplement, N2, and N-Acetyl-L-cysteine, growth factors R-spondin, mNoggin, EGF (called ENR), Y-27632 p160ROCK inhibitor. To promote cell proliferation CHIR and Valproic acid were added three days post embedding (collectively called CV). Medium was replenished after 2 days of culture. After three days of proliferation, medium was changed to a differentiation growth medium (ENR only). Organoid sampling  performed three days after culturing in ENRCV condition, and during three days of differentiation in ENR.

extract_protocol:
Single organoids collected by dissolving Matrigel with cold Cell Recovery Solution, followed by hand-picking and transferring to Nunc microwell culture plate with a dissociation mix containing protease isolated from B. licheniformis, EDTA, EGTA, DNase I, Accutase and PBS. Single organoids were dissociated by combining trituration using a siliconized pipette tips every 5 minutes and incubation at 37oC on for 15 minutes total. Following the dissociation, cell suspensions were diluted cell loading buffer in the loading tip of the DisCo chip.
After bead-cell in droplet co-encapsulation, the gel loading tip containing the sample droplets was transferred to a bead collection chip inlet (cp-chip; as described in Lab on a chip). Droplets in the tip were flushed to bead collection chip. Subsequently to bead capture, washing was performed as in Drop-seq with SSC and Reverse transcription buffer directly in the recovery chip. Reverse transcription solution was added to the beads and the recovery chip was placed on a heating block to perform first strand cDNA synthesis (RT) for 90 minutes at 42oC. After RT reaction beads were washed on the recovery chip with TE-SDS once, with TE-TW twice and with Tris once. The beads were treated with Exonuclease I for 45 minutes at 37oC to remove single-stranded oligonucleotides on beads. After Exonuclease I treatment, beads were washed with TE-SDS once, with TE-TW twice (as after reverse transcription). Beads were then eluted from the recovery chip in dH2O. cDNA was amplified for 18 – 23 cycles using Kapa HiFi Hot start ready mix. cDNA was purified with CleanPCR magnetic beads using 0.6X ratio to remove small cDNA fragments and primers. To assess the cDNA yield and quality concentration was measured using Qubit and cDNA traces quality was assessed using Fragment Analyzer (Agilent). cDNA was tagmented with in-house Tn5 (generated as described in Picelli et al..) and the library was purified using CleanPCR magnetic beads (0.6X ratio).
Libraries were sequenced on a NextSeq 500 system (Illumina) following recommendations from original protocol (20 bp for read 1 and 50 bp for read2).

treatment_protocol: After matrigel polymerization, cells were cultured in DMEM/F12 supplemented with Glutamine, Hepes, penicillin-streptomycin, B27 supplement, N2, and N-Acetyl-L-cysteine, growth factors R-spondin, mNoggin, EGF (called ENR), Y-27632 p160ROCK inhibitor. To promote cell proliferation CHIR and Valproic acid (collectively called CV) were added three days post embedding. Medium was replenished after 2 days of culture. After three days of proliferation, medium was changed to a differentiation growth medium (ENR only). Organoid sampling  performed three days after culturing in ENR CV condition, and during three days of differentiation in ENR.

value: Ex vivo cell culture

tag: sample day

value: Intestinal organoid


title: S3d


title: S3f


title: S3e


title: N_7068_het_sc


title: A471_het_SC


title: S3b


title: S3a


title: S2f


title: S2e


title: S2g


title: S2d


title: S2b


title: S2c


title: S2a


title: S1h


title: S1g


title: S1f


title: S1d


title: S1c


title: S3g


title: S1b


title: S0h


title: S3c


title: S0f


title: S0g


title: S0e


title: S0d


title: Δmas-infected 16 hours_3

first: Amy

middle: K.

last: Barczak

state: MA

address: The Ragon Institute, 400 Technology Square

department: Medicine/Infectious Diseases

institute: Massachusetts General Hospital

data_processing:
Libraries passing QC metrics were sequenced on an Illumina NextSeq 500 device with a NextSeq® 500/550 High Output Kit v2 (75 cycles)
35 base paired end reads were performed.
Sequences were trimmed with Trimomatic
Alignment was performed using Tophat 2.1 in GenePattern.
FPKM were calculated using Cufflinks (2.0.2) in GenePattern.
Genome_build: GRCm38
Supplementary_files_format_and_content: FPKM values for each sample

source_name: Bone marrow-derived macrophages

growth_protocol: C57BL6 bone marrow-derived macrophages were plated in media (DMEM with 20% FBS and 25ng/mL M-CSF) 1 day prior to infection. Each Mtb strain was grown to mid-log phase, washed in PBS, and resuspended in PBS prior to infection of macrophages.

extract_protocol:
RNA was harvested in RLT Plus buffer (Qiagen) and extracted using an Rneasy kit (Qiagen) according to the manufacturer's protocol.
Libraries were constructed using an NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina® and NEBNext Poly(A) mRNA magnetic isolation module (NEB #E7490) according to the manufacturer's protocol. Briefly, after mRNA enrichment, mRNAs were fragmented and cDNA was synthesised using random primers and the directional library was constructed using manufacturer's protocol.

treatment_protocol: BMDM were infected with each Mtb strain at an MOI of 2:1.


title: S0c


title: S0b


title: Δmas-infected 16 hours_2


title: H37Rv-infected 16 hours_3


title: H37Rv-infected 16 hours_2


title: Δmas-infected 16 hours_1


title: Tn::eccCa1-infected 16 hours_1


title: S0a


title: H37Rv-infected 16 hours_1


title: 4 M2+DMSO

city: Birmingham

first: Dongquan

last: Chen

state: AL

address: 1717 11th Ave South

department: Medicine

institute: Univ of Alabama at Birmingham

description:
macrophage_results-gene_reads.txt
macrophage_results-gene_reads_rpkm.txt

data_processing:
Sequence reads in fastq format were trimmed at each end of reads using read quality score vendor sequencing adaptors removed, and reads aligned against human genome hg19 using STAR algorithm
Adaptor sequences were removed using Cutadapt tool before alignment HISAT2 algorithm.  Reads Per Kilobase of transcript per million mapped reads (RPKM) methods were  applied for normalization after PCR replicates were removed using SAMtools.  Gene level quantification and statistics were performed using Partek Genomic Suite (PGS, St Louis, MO).
Genome_build: mm10

source_name: Macrophages

growth_protocol: RAW 264.7 cells were grown in DMEM supplemented with 10% FBS. Cells were polarized to the M2 phenotype by adding 20ng/mL M-CSF and 10ng/mL IL-4 for 24hours.

extract_protocol:
RNA from cells was harvested using Qiagen RNeasy Mini Kit (Valencia, CA) according to protocol
RNA libraries were prepared for sequencing using standard Illumina protocols

treatment_protocol: Cells (RAW 264.7) were treated with M-Csf (20 ng/ml) overnight followed by DMSO or GANT61 (10 μM) four hours prior to treatment with IL-4 (10 ng/ml) for 15 minutes.

value: RAW 264.7 Cells

value: DMSO


title: Δmas-infected 12 hours_2


title: S1a


title: Sample_ED_7

city: Santiago

first: Juan

last: Larraín

address: Portugal 49

department: Cellular and Molecular Biology

country: Chile

institute: Pontificia Universidad Catolica de Chile

data_processing:
Illumina Casava 1.7 software was used for basecalling.
Paired-end reads were mapped to Xenopus laevis model transcriptome v9.1 (Xenbase) using Bowtie. Gene expression level was quantified by RSEM.

organism: Xenopus laevis

source_name: 7h - uninjured - S7

extract_protocol:
Spinal cords from animals at R-stage were dissected in time-series after surgery and immediately stored in RNAlater. Posterioly the purification of total RNA was performed using the RNeasy Mini Kit.
RNA libraries were prepared by TruSeq RNA Library Prep Kit v2. Posterioly these were sequenced on the Illumina HiSeq 2000 and HiSeq 4000.

value: Caudal section of spinal cord

tag: Stage

value: NF stage 50

tag: surgery

value: uninjured

tag: time of isolation after surgery (hours)


title: S1e

tag: culture method


title: Sample_ED_9

source_name: 9h - uninjured - S7


title: Sample_ED_8

source_name: 8h - uninjured - S7


title: Tn::eccCa1-infected 12 hours_2


title: Sample_ED_6

source_name: 6h - uninjured - S7


title: H37Rv-infected 12 hours_2


title: Δmas-infected 12 hours_1


title: H37Rv-infected 12 hours_1


title: Sample_ED_5

source_name: 5h - uninjured - S7


title: Δmas-infected 8 hours_3


title: Sample_ED_3

source_name: 3h - uninjured - S7


title: Δmas-infected 8 hours_2


title: Δmas-infected 8 hours_1


title: Sample_ED_21

source_name: 21h - uninjured - S8


title: 6 M2+GANT61

value: GANT61 (10 uM)


title: Sample_ED_4

source_name: 4h - uninjured - S7


title: Tn::eccCa1-infected 8 hours_3


title: Sample_ED_2

source_name: 2h - uninjured - S7


title: Sample_ED_19

source_name: 19h - uninjured - S8


title: Sample_ED_20

source_name: 20h - uninjured - S8


title: Tn::eccCa1-infected 8 hours_2


title: Sample_ED_16

source_name: 16h - uninjured - S8


title: Sample_ED_18

source_name: 18h - uninjured - S8


title: H37Rv-infected 8 hours_1


title: Tn::eccCa1-infected 12 hours_1


title: Tn::eccCa1-infected 8 hours_1


title: Δmas-infected 4 hours_3


title: Sample_ED_15

source_name: 15h - uninjured - S8


title: H37Rv-infected 8 hours_2


title: Δmas-infected 4 hours_1


title: Sample_ED_14

source_name: 14h - uninjured - S8


title: Δmas-infected 4 hours_2

source_name: Bone marrow-derived macrophages


title: Tn::eccCa1-infected 4 hours_3


title: Sample_ED_13_1

source_name: 13h - uninjured - S7


title: Sample_ED_13_2

source_name: 13h - uninjured - S8


title: H37Rv-infected 4 hours_2


title: Tn::eccCa1-infected 4 hours_2


title: Sample_ED_12_1

source_name: 12h - uninjured - S7


title: Tn::eccCa1-infected 4 hours_1


title: Sample_ED_11

source_name: 11h - uninjured - S7


title: Sample_ED_17

source_name: 17h - uninjured - S8


title: H37Rv-infected 4 hours_1


title: Sample_ED_1

source_name: 1h - uninjured - S7


title: Uninfected_3


title: Sample_ED_10

source_name: 10h - uninjured - S7


title: Sample_ED_0_2

source_name: 0h - uninjured - S8


title: Sample_ED_21_TRANS

source_name: 21h - transection - S6

value: transection


title: Sample_ED_21_SHAM

source_name: 21h - sham - S6'

value: sham


title: Sample_ED_20_TRANS

source_name: 20h - transection - S6


title: Sample_ED_19_TRANS

source_name: 19h - transection - S6


title: Sample_ED_19_SHAM

source_name: 19h - sham - S6'


title: Sample_ED_12_2

source_name: 12h - uninjured - S8


title: Uninfected_1


title: Sample_ED_20_SHAM

source_name: 20h - sham - S6'


title: Sample_ED_18_TRANS

source_name: 18h - transection - S6


title: Sample_ED_18_SHAM

source_name: 18h - sham - S6'


title: Sample_ED_17_TRANS

source_name: 17h - transection - S6


title: Sample_ED_17_SHAM

source_name: 17h - sham - S6'


title: Colonic cells, Dok3-/- genotype, six days after DSS treatment, biological replicate 4

type: RNA

city: Singapore

first: Alison

middle: P

last: Lee

address: 20 Biopolis Way

country: Singapore

institute: Bioprocessing Technology Institute

description: Gene expression data from colonic cells of Dok3-deficient mice six days after DSS treatment.

hyb_protocol: According to Affymetrix protocol.

scan_protocol: According to Affymetrix protocol.

data_processing: Raw data from CEL files was RMA-normalized and summarized to transcript clusters using Partek Genomics Suite.

label: biotin

source_name: Colon, Dok3-/- genotype, six days after DSS treatment

label_protocol: Expression profiling was carried out on Affymetrix GeneChip Mouse Gene 2.0 ST Array according to manufacturer's protocol.

growth_protocol: C57BL/6 mice were purchased from The Jackson Laboratory. Dok3-/- mice were generated as described previously (Ng, Xu and Lam, 2007). Dok3-/- mice were backcrossed to C57BL/6 mice for more than 10 generations. Male and female mice were used at 8 to 10 weeks of age unless otherwise stated. All mice were maintained under specific pathogen-free conditions at A*STAR Biological Resource Centre (BRC).

extract_protocol: Total RNA was extracted using TRIzol (Invitrogen) and precipitated with isopropanol, followed by DNase I digestion using RNase-Free DNase Set and purification using RNeasy MinElute Cleanup Kit (both kits from Qiagen).

treatment_protocol: Mice were treated with 2% (w/v) DSS (Sigma-Aldrich) dissolved in sterile, distilled water ad libitum for 7 days followed by normal drinking water till end of experiment.

tag: genotype

value: colonic cells

value: six days after DSS


title: Sample_ED_16_TRANS

source_name: 16h - transection - S6


title: Sample_ED_0_1

source_name: 0h - uninjured - S7


title: Uninfected_2

source_name: Bone marrow-derived macrophages


title: Sample_ED_0_T_S

source_name: 0h - uninjured - S6,S6'


title: Sample_ED_16_SHAM

source_name: 16h - sham - S6'


title: Colonic cells, Dok3-/- genotype, six days after DSS treatment, biological replicate 1


title: Colonic cells, Dok3-/- genotype, six days after DSS treatment, biological replicate 2


title: Sample_ED_18h_Trans

source_name: 18h - transection - S5


title: Colonic cells, WT genotype, six days after DSS treatment, biological replicate 2

description: Gene expression data from colonic cells of WT mice six days after DSS treatment.

source_name: Colon, WT genotype, six days after DSS treatment


title: Colonic cells, WT genotype, six days after DSS treatment, biological replicate 1


title: Colonic cells, Dok3-/- genotype, before DSS treatment, biological replicate 2

description: Gene expression data from colonic cells of Dok3-deficient mice before DSS treatment.

source_name: Colon, Dok3-/- genotype, before DSS treatment

value: before DSS


title: Colonic cells, Dok3-/- genotype, before DSS treatment, biological replicate 3


title: Sample_ED_18h_Sham

source_name: 18h - sham - S5'


title: Colonic cells, WT genotype, six days after DSS treatment, biological replicate 3


title: Colonic cells, WT genotype, six days after DSS treatment, biological replicate 4


title: Colonic cells, WT genotype, before DSS treatment, biological replicate 4

description: Gene expression data from colonic cells of WT mice before DSS treatment.

source_name: Colon, WT genotype, before DSS treatment


title: sh_NC

city: Nanjing

first: chen

last: Wang

address: xianlin street num 163

country: China

institute: Nanjing University

description: control cell

data_processing:
For the data analysis, base calls are performed using CASAVA.
Sequencing tags were aligned to the human genome using TopHat.
Cufflinks was used to calculate the fragments per kilobase of transcript sequence per million base pairs sequenced (FPKM). Differentially expressed genes with a p-value of 0.005 or less and an absolute fold change of 2 or more were clustered and visualized using a clustergram heatmap.
For enrichment analysis of Gene Ontology (GO) and KEGG (Kyoto Encyclopedia of Genes and Genomes), the differentially expressed gene lists were analyzed in DAVID (The Database for Annotation, Visualization and Integrated Discovery annotation tools [http://david.abcc.ncifcrf.gov/]) and selected based on p < 0.01.
Genome_build: mm9
Supplementary_files_format_and_content: Matrix table with raw gene counts for every gene and every sample
Supplementary_files_format_and_content: tab-delimited text file include FPKM values for each Sample ...

source_name: Human colorectal carcinoma cells

extract_protocol:
For transcriptome sequencing analysis, total RNA was extracted from HCT116 cells stably expressing or inhibiting CBFβ (CBFβ-LV/shRNA-CBFβ-LV groups) or their control cells (Control-LV/shRNA-Control-LV groups) using TRIzol (Life Technologies). All of the procedures were carried out by Novogene (Beijing, China) according to Illumina’s protocols. RNA was subjected to fragmentation and library construction and then sequenced with the HiSeq 2000 platform.
RNA libraries were prepared for sequencing using standard Illumina protocols

value: shRNA-Control-LV-HCT116 cells


title: Sample_ED_17h_Sham

source_name: 17h - sham - S5'


title: Sample_ED_17h_Trans

source_name: 17h - transection - S5


title: Sample_ED_16h_Trans

source_name: 16h - transection - S5


title: NC

description: control cell

value: Control-LV-HCT116 cells


title: sh_CBFB

description: inhibit expression of CBFβ

value: shRNA-CBF[beta]-LV-HCT116 cells


title: CBFB

description: stably express CBFβ

value: CBF[beta]-LV-HCT116 cells


title: Sample_ED_15h_Sham

source_name: 15h - sham - S5'


title: H_3_1

city: Jinan

first: Yan

last: Zheng

state: Shandong

address: No.440 Jiyan road

department: Shandong Provincial Key Laboratory of Radiation Oncology

institute: Shandong Cancer Hospital Affiliated to Shandong University

data_processing:
Illumina Casava1.7 software used for basecalling.
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence, then mapped to whole transcriptome using tophat2 with default parameters.
Reads count of three samples were calculated by HT-Seq based on the gtf file of ensembl human.
Genome_build: hg19
Supplementary_files_format_and_content: tab-delimited text files include gene count values for each Sample

source_name: HGC-27-transfection - pcDNA3.1

growth_protocol: Human HGC-27 cells were cultured in RPMI-1640 media supplied with 10% fetal bovine serum (Hyclone) at 37°C with 5% CO2.

extract_protocol: Trizol extraction of total RNA was performed according to the manufacturer's instructions.

treatment_protocol: lncRNA lncPSCA expression plasmid pcDNA3.1-lncPSCA or pcDNA3.1 vector were transfected into HGC-27 cells with INTERFERin® (Polyplus). Fourty-eight hours later, cells were collected.

value: gastric adenocarcinoma

tag: cell line

value: control


title: Sample_ED_14h_Trans

source_name: 14h - transection - S5


title: Colonic cells, Dok3-/- genotype, six days after DSS treatment, biological replicate 3

source_name: Colon, Dok3-/- genotype, six days after DSS treatment


title: Colonic cells, Dok3-/- genotype, before DSS treatment, biological replicate 1


title: Sample_ED_13h_Sham

source_name: 13h - sham - S5'


title: H_lncPSCA

source_name: HGC-27-transfection - pcDNA3.1-lncPSCA

value: lncPSCA overexpression


title: Sample_ED_16h_Sham

source_name: 16h - sham - S5'


title: miR_Tst_3

city: Changchun

first: Boyin

last: Jia

state: Jilin

address: 2888 Xincheng Street

department: College of Animal Science and Technology

institute: Jilin Agricultural University

data_processing:
CASAVA  bcl2fastq-1.8.4 used for basecalling.
The clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq SR Cluster Kit v3-cBot-HS (Illumia) according to the manufacturer’s instructions. After cluster generation, the library preparations were sequenced on an Illumina Hiseq 2500/2000 platform and 50bp single-end reads were generated.
Raw data (raw reads) of fastq format were firstly processed through custom perl and python scripts. In this step, clean datas(clean reads) were obtained by removing reads containing ploy-N, with 5’ adapter contaminants, without 3’ adapter or the insert tag, containing ploy A or T or G or C and low quality reads from raw data. At the same time, Q20, Q30, and GC-content of the raw datas were calculated. Then, chose a certain range of length from clean reads to do all the downstream analyses. The small RNA tags were mapped to reference sequence by Bowtie without mismatch to analyze their expression and distribution on the reference.
Genome_build: reference sequence used for read alignment
Supplementary_files_format_and_content: tab-delimited text files include TPM values for each Sample

organism: Cervus nippon

source_name: testis

extract_protocol:
Testes were removed, flash frozen on dry ice, and RNA was harvested using Trizol reagent. A total amount of 3 μg total RNA per sample was used as input material for the small RNA library. Sequencing libraries were generated using NEBNext®Multiplex Small RNA Library Prep Set for Illumina® (NEB, USA.) following manufacturer’s recommendations and index codes were added to attribute sequences to each sample.
the small RNA libraries were prepared for sequencing using standard Illumina protocols

tag: breed

value: Shuangyang

tag: tissus

value: testis

tag: developmental stage


title: miR_Tst_4


title: miR_Tst_2


title: Sample_ED_13h_Trans

source_name: 13h - transection - S5

tag: tissue

value: Caudal section of spinal cord

value: NF stage 50


title: Sample_ED_14h_Sham

source_name: 14h - sham - S5'


title: Sample_ED_12h_Sham

source_name: 12h - sham - S5'


title: Sample_ED_12h_Trans

source_name: 12h - transection - S5


title: miR_Tst_1


title: Sample_ED_15h_Trans

source_name: 15h - transection - S5


title: Sample_ED_11h_Sham

source_name: 11h - sham - S5'


title: Tst_4

data_processing:
The clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumia) according to the manufacturer’s instructions. After cluster generation, the library preparations were sequenced on an Illumina Hiseq 2500 platform and paired-end reads were generated.
Raw data (raw reads) of fastq format were firstly processed through in-houseperl 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, GC-content and sequence duplication level of the clean data were calculated. All the downstream analyses were based on clean data with high quality.
The left files (read1 files) from all libraries/samples were pooled into one big left.fq file, and right files (read2 files) into one big right.fq file. Transcriptome assembly was accomplished based on the left.fq and right.fq using Trinity (Grabherr et al, 2011) with min_kmer_cov set to 2 by default and all other parameters set default.
Genome_build: de novo sequencing without reference genome
Supplementary_files_format_and_content: tab-delimited text files include FPKM values for each Sample

extract_protocol:
Testes  were removed, flash frozen on dry ice, and RNA was harvested using Trizol reagent. Illumina TruSeq RNA Sample Prep Kit (Cat#FC-122-1001) was used with 1 ug of total RNA for the construction of sequencing libraries.
A total amount of 3 μg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer’s recommendations and index codes were added to attribute sequences to each sample. 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 NEBNext First Strand Synthesis Reaction Buffer（5X）. First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase（RNaseH- Clustering and sequencing (Novogene Experimental Department)）. Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. In the reaction buffer, dNTPs with dTTPwere replaced by dUTP. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3’ ends of DNA fragments, NEBNext Adaptor with hairpin loop structurewere ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 150~200 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Then 3 μlUSER Enzyme (NEB， USA) 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, products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100system.

tag: age


title: TET_EXP20001394_Sample_06_hashed sample

city: Boston

first: Natalie

middle: Y. R.

last: Agar

state: Massachusetts

address: Harvard Medical School

department: Neurosurgery

institute: Brigham and Women's Hospital

description: TET

data_processing:
Base Calling TCR BCL to TCR FASTQ via Cell Ranger 3.1.0 with the flag "--delete-undetermined" in addition to the standard settings.
Base Calling GEX BCL to GEX FASTQ via Cell Ranger 3.1.0 with the flag "--delete-undetermined" in addition to the standard settings.
Base Calling ADT BCL to ADT FASTQ via Cell Ranger 3.1.0 with the flag "--delete-undetermined" in addition to the standard settings.
Base Calling tetramer BCL to tetramer FASTQ file via bcl2fastq2 version v2.20.0 with the flags "--minimum-trimmed-read-length 0 --mask-short-adapter-reads 0" in addition to the standard settings.
TCR Read Counts: FASTQ to vdj counts via Cell Ranger 3.1.0 using the standard settings.
GEX Read Counts: FASTQ to gene expression counts via Cell Ranger 3.1.0 using the standard settings.
Tetramer FASTQ to Tetramer counts via extracting tetramer barcode from uniquely observed tetramer UMI reads using python script
ADT FASTQ to ADT counts via extracting tetramer barcode from uniquely observed tetramer UMI reads using python script
TCR VDJ counts paired with tetramer and ADT counts via common hydrogel barcode using python script
VDJ and tetramer counts separated via hashtags using Seurat 4.0.3
Dehashed VDJs selected for functional assays.
Genome_build: GRCh38 (hg19)
Supplementary_files_format_and_content: The processed data files conform to the hfad AnnData format: https://anndata.readthedocs.io/en/latest/anndata.AnnData.html
Supplementary_files_format_and_content: Metadta for hashed samples including HTO oligo sequence is available in the file "GEO_upload_seq_CIPHER_2021_11_04_Supplemental_Sample_to_HTO.xlsx"

source_name: CD8+ T cells

extract_protocol:
PBMCs were thawed, and CD8+ T cells were enriched by magnetic-activated cell sorting (MACS) using a CD8+ T Cell Isolation Kit (Miltenyi) following the manufacturers protocol. The CD8+ T cells were then stained with 1 nM final concentration tetramer library in the presence of 2 mg/mL salmon sperm DNA in PBS with 0.5% BSA solution for 20 minutes. Cells were then labeled with anti-TCR antibody-derived tag (ADT, IP26, Biolegend) for 15 minutes followed by washing. Tetramer bound cells were then labeled with phycoerythrin (PE) conjugated anti-DKDDDDK-Flag antibody (BioLegend) followed by dead cell discrimination using 7-amino-actinomycin D (7-AAD). The live, tetramer positive cells were sorted using a Sony MA900 Sorter (Sony). Tetramer positive cells were counted by Nexcelom Cellometer (Lawrence, MA, USA) using AOPI stain following manufacturer’s recommended conditions. Single-cell encapsulations were generated utilizing 5’ v1 Gem beads from 10x Genomics (Pleasanton, CA, USA) on a 10x Chromium controller and downstream TCR, and Surface marker libraries were made following manufacturer recommended conditions. All libraries were quantified on a BioRad CFX 384 (Hercules, CA, USA) using Kapa Biosystems (Wilmington, MA, USA) library quantified kits and pooled at an equimolar ratio. TCRs, surface markers, and tetramer generated libraries were sequenced on Illumina (San Diego, CA, USA) NextSeq550 instruments. Sequencing data were processed using the Cell Ranger Software Suite (Version 3). Samples were demultiplexed and unique molecular identifier (UMI) counts were quantified for TCRs, tetramers, and gene expression.
10X Chromium 5' single-cell

value: PBMC

tag: subject

value: multiplexed sample

tag: cohort


title: TET_EXP20001394_Sample_04_hashed sample

description: TET


title: Tst_3


title: Tst_2


title: TET_EXP20001394_Sample_05_hashed sample

description: TET


title: Tst_1


title: Sample_ED_10h_Sham

source_name: 10h - sham - S5'

value: sham


title: Sample_ED_0h

source_name: 0h - uninjured - S5,S5'

value: uninjured


title: Sample_9T2

source_name: 9h - transection - S4

value: transection


title: Colonic cells, WT genotype, before DSS treatment, biological replicate 2

source_name: Colon, WT genotype, before DSS treatment

tag: strain

value: WT

value: colonic cells

value: before DSS


title: Sample_9S2

source_name: 9h - sham - S4'

tag: treatment


title: Sample_8T2

source_name: 8h - transection - S4


title: Sample_ED_11h_Trans

source_name: 11h - transection - S5


title: Sample_8S2

source_name: 8h - sham - S4'


title: TET_EXP20001394_Sample_01_hashed sample

description: TET

tag: cell type


title: TET_EXP20001394_Sample_03_hashed sample

description: TET


title: Sample_7S2

source_name: 7h - sham - S4'


title: Sample_6T2

source_name: 6h - transection - S4


title: Sample_ED_10h_Trans

source_name: 10h - transection - S5


title: TET_EXP20001394_Sample_02_hashed sample

description: TET


title: TET_EXP20001260_Sample_05_hashed sample

description: TET


title: Sample_6T1

source_name: 6h - transection - S3


title: TET_EXP20001265_Sample_02_hashed sample

description: TET


title: Colonic cells, Dok3-/- genotype, before DSS treatment, biological replicate 4

source_name: Colon, Dok3-/- genotype, before DSS treatment


title: TET_EXP20001265_Sample_01_hashed sample

description: TET


title: TET_EXP20001260_Sample_04_hashed sample

description: TET


title: Sample_6S2

source_name: 6h - sham - S4'


title: Sample_6S1

source_name: 6h - sham - S3'


title: Sample_5T1

source_name: 5h - transection - S3


title: Sample_4T1

source_name: 4h - transection - S3


title: Sample_5S1

source_name: 5h - sham - S3'


title: Sample_7T2

source_name: 7h - transection - S4


title: TET_EXP20001260_Sample_03_hashed sample

description: TET

source_name: CD8+ T cells


title: TET_EXP20001249_Sample_06_hashed sample

description: TET


title: TET_EXP20001265_Sample_03_hashed sample

description: TET


title: TET_EXP20001249_Sample_05_hashed sample

description: TET


title: Sample_3T1

source_name: 3h - transection - S3


title: Sample_2S1

source_name: 2h - sham - S3'


title: Sample_3S1

source_name: 3h - sham - S3'


title: Sample_1S1

source_name: 1h - sham - S3'


title: Sample_2T1

source_name: 2h - transection - S3


title: Sample_12T2

source_name: 12h - transection - S4


title: Sample_4S1

source_name: 4h - sham - S3'


title: Sample_1T1

source_name: 1h - transection - S3


title: TET_EXP20001260_Sample_02_hashed sample

description: TET


title: TET_EXP20001234_Sample_01_hashed sample

description: TET


title: TET_EXP20001234_Sample_03_hashed sample

description: TET


title: TET_EXP20001249_Sample_03_hashed sample

description: TET


title: TET_EXP20001249_Sample_01_hashed sample

description: TET


title: TET_EXP20001234_Sample_04_hashed sample

description: TET


title: TET_EXP20001249_Sample_02_hashed sample

description: TET


title: TET_EXP20001249_Sample_04_hashed sample

description: TET


title: Sample_12S2

source_name: 12h - sham - S4'


title: TET_EXP20001234_Sample_02_hashed sample

description: TET


title: Sample_11T2

source_name: 11h - transection - S4


title: TET_EXP20001260_Sample_01_hashed sample

description: TET


title: TET_EXP20001148_Sample_03_hashed sample

description: TET


title: Sample_11S2

source_name: 11h - sham - S4'


title: TET_EXP20001148_Sample_02_hashed sample

description: TET


title: Sample_10T2

source_name: 10h - transection - S4


title: TET_EXP20001148_Sample_01_hashed sample

description: TET


title: Sample_10S2

source_name: 10h - sham - S4'


title: TET_EXP20001114_Sample_08

description: TET

value: unexposed


title: Colonic cells, WT genotype, before DSS treatment, biological replicate 3

source_name: Colon, WT genotype, before DSS treatment


title: Sample_0_2

source_name: 0h - uninjured - S4,S4'


title: TET_EXP20001114_Sample_07

description: TET


title: TET_EXP20001114_Sample_06

description: TET


title: Sample_90TRANS_ED23

source_name: 1.5h - transection - S2


title: TET_EXP20001148_Sample_04_hashed sample

description: TET

source_name: CD8+ T cells


title: Sample_90SHAM_ED23


title: Sample_90TRANS_ED20

source_name: 1.5h - transection - S1


title: Colonic cells, WT genotype, before DSS treatment, biological replicate 1


title: TET_EXP20001114_Sample_04

description: TET


title: Sample_90SHAM_ED20


title: Sample_60TRANS_ED20

source_name: 1h - transection - S1


title: TET_EXP20001114_Sample_05

description: TET


title: Sample_60SHAM_ED23

source_name: 1h - sham - S2'


title: TET_EXP20001114_Sample_02

description: TET


title: Sample_60TRANS_ED23

source_name: 1h - transection - S2


title: TET_EXP20001095_Sample_06

description: TET


title: TET_EXP20001114_Sample_01

description: TET


title: TET_EXP20001114_Sample_03

description: TET


title: Sample_50_2_ED23

source_name: 0h - uninjured - S2,S2'


title: TET_EXP20001095_Sample_07

description: TET

value: convalescent


title: TET_EXP20001095_Sample_08

description: TET


title: Sample_60SHAM_ED20

source_name: 1h - sham - S1'


title: Sample_50_2_ED20

source_name: 0h - uninjured - S1,S1'


title: TET_EXP20001095_Sample_04

description: TET


title: TET_EXP20001095_Sample_05

description: TET


title: Sample_50_1_ED23


title: Sample_50_1_ED20


title: Sample_0_1

source_name: 0h - uninjured - S3,S3'


title: TET_EXP20001095_Sample_02

description: TET


title: TET_EXP20001095_Sample_03

description: TET


title: Sample_30TRANS_ED23

source_name: 0.5h - transection - S2


title: Sample_30TRANS_ED20

source_name: 0.5h - transection - S1


title: TET_EXP20001003_Sample_08

description: TET


title: TET_EXP20001095_Sample_01

description: TET


title: TET_EXP20001003_Sample_06

description: TET


title: TET_EXP20001003_Sample_07

description: TET


title: Sample_30SHAM_ED23


title: Sample_30SHAM_ED20


title: Sample_120TRANS_ED23

source_name: 2h - transection - S2


title: Sample_120SHAM_ED23

source_name: 2h - sham - S2'


title: Sample_120TRANS_ED20

source_name: 2h - transection - S1


title: Sample_120SHAM_ED20

source_name: 2h - sham - S1'


title: TET_EXP20001003_Sample_01

description: TET


title: TET_EXP20001003_Sample_05

description: TET


title: TET_EXP20000910_Sample_09

description: TET


title: TET_EXP20001003_Sample_04

description: TET


title: TET_EXP20000910_Sample_11

description: TET


title: TET_EXP20000910_Sample_10

description: TET


title: TET_EXP20001003_Sample_02

description: TET


title: TET_EXP20000910_Sample_12

description: TET


title: TET_EXP20000910_Sample_08

description: TET


title: TET_EXP20000910_Sample_07

description: TET


title: PBMC-health-rep4

city: Chengdu

first: Fangyuan

last: Shen

address: No.29 Wangjiang Road

institute: Sichuan University

description:
Gene expression of healthy control
D26_NS

hyb_protocol: Each slide was hybridized with 100ng Cy3‐labeled RNA using miRNA Complete  Labeling and Hyb Kit(Cat#5190‐0456, Agilent technologies, Santa Clara, CA, US)in  hybridization Oven(Cat#G2545A, Agilent technologies, Santa Clara, CA, US)at 55℃， 20rpm  for  20  hours  according  to  the  manufacturer’s  instructions,  hybridization  section. After hybridization, slides were washed in staining dishes (Cat#121, Thermo  Shandon, Waltham, MA, US) with Gene Expression Wash Buffer Kit(Cat#5188‐5327,  Agilent technologies, Santa Clara, CA, US).

scan_protocol: Slides  were  scanned  by  Agilent  Microarray  Scanner(Cat#G2565BA，Agilent  technologies,  Santa  Clara,  CA,  US)and  Feature  Extraction  software  10.7  (Agilent  technologies, Santa Clara, CA, US)with default settings.

source_name: PBMCs, health, replicate 4

label_protocol: miRNA molecular in total RNA was labeled by miRNA Complete Labeling and Hyb  Kit(Cat#5190‐0456,  Agilent  technologies,  Santa  Clara,  CA,  US)followed  the  manufacturer’s instructions, labeling section.

tag: disease state

value: healthy

tag: gender

value: unknown

value: Peripheral blood mononuclear cells


title: PBMC-health-rep2

description:
Gene expression of healthy control
D30_NS

source_name: PBMCs, health, replicate 6


title: TET_EXP20000910_Sample_06

description: TET

source_name: CD8+ T cells


title: TET_EXP20000910_Sample_05

description: TET


title: TET_EXP20000910_Sample_04

description: TET


title: PBMC-health-rep6

description:
Gene expression of healthy control
D11_NS

source_name: PBMCs, health, replicate 2


title: TET_EXP20000910_Sample_03

description: TET


title: PBMC-AIDS-rep50

description:
Gene expression of AIDS patient after ART
B9_NS

source_name: PBMCs, AIDS, replicate 50

value: AIDS

value: male


title: PBMC-health-rep7

description:
Gene expression of healthy control
D08_NS

source_name: PBMCs, health, replicate 1


title: PBMC-health-rep5

description:
Gene expression of healthy control
D23_NS

source_name: PBMCs, health, replicate 3


title: TET_EXP20000910_Sample_02

description: TET


title: PBMC-health-rep3

description:
Gene expression of healthy control
D29_NS

source_name: PBMCs, health, replicate 5


title: PBMC-health-rep1

description:
Gene expression of healthy control
D31_NS

source_name: PBMCs, health, replicate 7


title: TET_EXP20000910_Sample_01

description: TET


title: PBMC-AIDS-rep48

description:
Gene expression of AIDS patient after ART
B7_NS

source_name: PBMCs, AIDS, replicate 48

tag: virus load

value: LVL


title: TET_EXP20000880_Sample_04_hashed sample

description: TET


title: PBMC-AIDS-rep49

description:
Gene expression of AIDS patient after ART
B8_NS

source_name: PBMCs, AIDS, replicate 49


title: PBMC-AIDS-rep46

description:
Gene expression of AIDS patient after ART
B5_NS

source_name: PBMCs, AIDS, replicate 46

value: HVL


title: PBMC-AIDS-rep47

description:
Gene expression of AIDS patient after ART
B6_NS

source_name: PBMCs, AIDS, replicate 47


title: TET_EXP20000880_Sample_03_hashed sample

description: TET

value: PBMC


title: TET_EXP20000880_Sample_01_hashed sample

description: TET

value: multiplexed sample


title: TET_EXP20000854_Sample_08

description: TET


title: TET_EXP20000854_Sample_07

description: TET


title: TET_EXP20000854_Sample_05

description: TET


title: TET_EXP20000854_Sample_06

description: TET


title: PBMC-AIDS-rep45

description:
Gene expression of AIDS patient after ART
B4_NS

source_name: PBMCs, AIDS, replicate 45


title: TET_EXP20000854_Sample_01

description: TET


title: TET_EXP20000854_Sample_04

description: TET


title: PBMC-AIDS-rep43

description:
Gene expression of AIDS patient after ART
B29_NS

source_name: PBMCs, AIDS, replicate 43


title: PBMC-AIDS-rep41

description:
Gene expression of AIDS patient after ART
B26_NS

source_name: PBMCs, AIDS, replicate 41


title: PBMC-AIDS-rep42

description:
Gene expression of AIDS patient after ART
B28_NS

source_name: PBMCs, AIDS, replicate 42


title: PBMC-AIDS-rep39

description:
Gene expression of AIDS patient after ART
B24_NS

source_name: PBMCs, AIDS, replicate 39


title: TET_EXP20000880_Sample_02_hashed sample

description: TET


title: PBMC-AIDS-rep38

description:
Gene expression of AIDS patient after ART
B22_NS

source_name: PBMCs, AIDS, replicate 38


title: PBMC-AIDS-rep44

description:
Gene expression of AIDS patient after ART
B3_NS

source_name: PBMCs, AIDS, replicate 44


title: PBMC-AIDS-rep37

description:
Gene expression of AIDS patient after ART
B21_NS

source_name: PBMCs, AIDS, replicate 37


title: PBMC-AIDS-rep34

description:
Gene expression of AIDS patient after ART
B18_NS

source_name: PBMCs, AIDS, replicate 34


title: PBMC-AIDS-rep35

description:
Gene expression of AIDS patient after ART
B19_NS

source_name: PBMCs, AIDS, replicate 35

value: female


title: TET_EXP20000768_Sample_04

description: TET

source_name: CD8+ T cells


title: TET_EXP20000768_Sample_03

description: TET


title: PBMC-AIDS-rep33

description:
Gene expression of AIDS patient after ART
B17_NS

source_name: PBMCs, AIDS, replicate 33


title: PBMC-AIDS-rep32

description:
Gene expression of AIDS patient after ART
B16_NS

source_name: PBMCs, AIDS, replicate 32


title: PBMC-AIDS-rep40

description:
Gene expression of AIDS patient after ART
B25_NS

source_name: PBMCs, AIDS, replicate 40


title: PBMC-AIDS-rep36

description:
Gene expression of AIDS patient after ART
B2_NS

source_name: PBMCs, AIDS, replicate 36


title: PBMC-AIDS-rep31

description:
Gene expression of AIDS patient after ART
B15_NS

source_name: PBMCs, AIDS, replicate 31


title: TET_EXP20000854_Sample_02

description: TET


title: TET_EXP20000780_Sample_03

description: TET


title: TET_EXP20000722_Sample_07

description: TET


title: TET_EXP20000722_Sample_06

description: TET


title: PBMC-AIDS-rep29

description:
Gene expression of AIDS patient after ART
B12_NS

source_name: PBMCs, AIDS, replicate 29


title: PBMC-AIDS-rep30

description:
Gene expression of AIDS patient after ART
B13_NS

source_name: PBMCs, AIDS, replicate 30


title: TET_EXP20000780_Sample_02

description: TET


title: TET_EXP20000722_Sample_04

description: TET


title: TET_EXP20000722_Sample_03

description: TET


title: TET_EXP20000722_Sample_01

description: TET


title: TET_EXP20000768_Sample_02

description: TET


title: PBMC-AIDS-rep27

description:
Gene expression of AIDS patient after ART
B10_NS

source_name: PBMCs, AIDS, replicate 27


title: TET_EXP20000854_Sample_03

description: TET


title: TET_EXP20000683_Sample_08

description: TET


title: PBMC-AIDS-rep24

description:
Gene expression of AIDS patient after ART
A6_NS

source_name: PBMCs, AIDS, replicate 24


title: PBMC-AIDS-rep25

description:
Gene expression of AIDS patient after ART
A7_NS

source_name: PBMCs, AIDS, replicate 25


title: TET_EXP20000683_Sample_01

description: TET


title: PBMC-AIDS-rep26

description:
Gene expression of AIDS patient after ART
B1_NS

source_name: PBMCs, AIDS, replicate 26


title: TET_EXP20000722_Sample_05

description: TET


title: TET_EXP20000683_Sample_07

description: TET


title: PBMC-AIDS-rep22

description:
Gene expression of AIDS patient after ART
A4_NS

source_name: PBMCs, AIDS, replicate 22


title: PBMC-AIDS-rep21

description:
Gene expression of AIDS patient after ART
A30_NS

source_name: PBMCs, AIDS, replicate 21


title: PBMC-AIDS-rep23

description:
Gene expression of AIDS patient after ART
A5_NS

source_name: PBMCs, AIDS, replicate 23


title: TET_EXP20000654_Sample_06

description: TET


title: PBMC-AIDS-rep20

description:
Gene expression of AIDS patient after ART
A3_NS

source_name: PBMCs, AIDS, replicate 20


title: TET_EXP20000654_Sample_05

description: TET


title: TET_EXP20000654_Sample_04

description: TET


title: PBMC-AIDS-rep19

description:
Gene expression of AIDS patient after ART
A28_NS

source_name: PBMCs, AIDS, replicate 19


title: TET_EXP20000654_Sample_03

description: TET

source_name: CD8+ T cells


title: TET_EXP20000654_Sample_02

description: TET


title: PBMC-AIDS-rep18

description:
Gene expression of AIDS patient after ART
A27_NS

source_name: PBMCs, AIDS, replicate 18


title: ADT_EXP20001394_Sample_06_hashed sample

description: ADT


title: PBMC-AIDS-rep17

description:
Gene expression of AIDS patient after ART
A26_NS

source_name: PBMCs, AIDS, replicate 17


title: PBMC-AIDS-rep16

description:
Gene expression of AIDS patient after ART
A25_NS

source_name: PBMCs, AIDS, replicate 16


title: ADT_EXP20001394_Sample_05_hashed sample

description: ADT


title: ADT_EXP20001394_Sample_04_hashed sample

description: ADT


title: ADT_EXP20001394_Sample_03_hashed sample

description: ADT


title: PBMC-AIDS-rep13

description:
Gene expression of AIDS patient after ART
A22_NS

source_name: PBMCs, AIDS, replicate 13


title: ADT_EXP20001394_Sample_02_hashed sample

description: ADT


title: PBMC-AIDS-rep14

description:
Gene expression of AIDS patient after ART
A23_NS

source_name: PBMCs, AIDS, replicate 14


title: ADT_EXP20001394_Sample_01_hashed sample

description: ADT


title: ADT_EXP20001265_Sample_02_hashed sample

description: ADT


title: PBMC-AIDS-rep12

description:
Gene expression of AIDS patient after ART
A21_NS

source_name: PBMCs, AIDS, replicate 12


title: ADT_EXP20001265_Sample_03_hashed sample

description: ADT


title: PBMC-AIDS-rep10

description:
Gene expression of AIDS patient after ART
A2_NS

source_name: PBMCs, AIDS, replicate 10


title: PBMC-AIDS-rep8

description:
Gene expression of AIDS patient after ART
A17_NS

source_name: PBMCs, AIDS, replicate 8


title: PBMC-AIDS-rep11

description:
Gene expression of AIDS patient after ART
A20_NS

source_name: PBMCs, AIDS, replicate 11


title: ADT_EXP20001265_Sample_01_hashed sample

description: ADT


title: PBMC-AIDS-rep5

description:
Gene expression of AIDS patient after ART
A14_NS

source_name: PBMCs, AIDS, replicate 5


title: ADT_EXP20001260_Sample_04_hashed sample

description: ADT


title: PBMC-AIDS-rep4

description:
Gene expression of AIDS patient after ART
A13_NS

source_name: PBMCs, AIDS, replicate 4


title: PBMC-AIDS-rep15

description:
Gene expression of AIDS patient after ART
A24_NS

source_name: PBMCs, AIDS, replicate 15


title: ADT_EXP20001260_Sample_03_hashed sample

description: ADT


title: ADT_EXP20001260_Sample_02_hashed sample

description: ADT


title: ADT_EXP20001260_Sample_01_hashed sample

description: ADT


title: ADT_EXP20001249_Sample_06_hashed sample

description: ADT


title: ADT_EXP20001249_Sample_05_hashed sample

description: ADT


title: D. melanogaster macrophages_DfosDN_Rep3

city: Klosterneuburg

first: Daria

last: Siekhaus

address: Am Campus 1

department: Bertalanffy Building

country: Austria

institute: IST Austria

description: FACS-sorted labled macrophages

data_processing:
Aligning the transcriptome sequence reads to the D. melanogaster reference genome (Ensembl BDGP6) with STAR version 2.5.1b using 2-pass alignment mode.
Counting the number of reads; the quality of the RNA-seq library was assessed by calculating the percentage of reads that are mapped  to exonic and intronic regions of the genome, while using polyA selection for library preparation.
the reads were associated with known genes and the number of reads aligned within each gene was counted using HTSeq tool version 0.5.4p3.
the counts were normalised using the TMM normalisation method of the edgeR R/Bioconductor package ( (R version 3.4.4, Bioconductor version 3.6)
The data were  log transformed using the voom approach in the limma package. For the visualizations and result files the TMM normalised counts are represented as TPM (Transcripts Per Kilobase Million) values.
Genome_build: the Drosophila melanogaster reference genome (Ensembl BDGP6)
Supplementary_files_format_and_content: tab-delimited text files include FPKM values for both wildtype and the mutant  samples
Supplementary_files_format_and_content: Matrix table with raw gene counts for both wildtype and the mutant  samples

organism: Drosophila melanogaster

source_name: Embryonic macrophages

growth_protocol: Fly embryos of both genotypes were collected for stage 11 at 29°C.

extract_protocol:
Total RNA was isolated from FACS-sorted mCherry-positive macrophages using Rneasy Plus Micro Kit (QIAGEN) and stored at -80C for quality control and RNA sequencing.
RNA libraries were prepared for sequencing using standard Illumina protocols (Illumina’s HiSeq2500, 50bp read length)

treatment_protocol: Dechorionated embryos were dissociated and Cherry-positive macrophages were sorted by FACS using the protocol from Attila Gyoergy et al., G3, 2018.

value: Embryonic macrophages from stage 11

value: Embryonic macrophages (hemocytes)

tag: tag

value: 3xMcherry-tagged macrophages


title: SPM

first: Marjan

last: Biočanin

address: Station 19, SV 3820

institute: EPFL

data_processing:
Drop-seq tools 2.3.0 package on the EPFL SCITAS HPC platform. After trimming and sequence tagging, reads were aligned to the mouse reference genome (mm10) using STAR (version 2.7.0.e). https://github.com/mccrowjp/Dropseq
Following the alignment, the gene annotation was added, bead synthesis errors were corrected and cell barcodes extracted. Subsequently, the BAM files containing the processed data were used to obtain digital gene expression matrices.
Downstream data analysis was carried out using R (version 3.5.0)
Genome_build: mm10-hg19 mixed genome GSE63269
Supplementary_files_format_and_content: tab-delimited text files include read-count matrices and digital gene expression files for each species

organism:
Homo sapiens
Mus musculus

source_name: HEK 293T/ murine brown preadipocyte line

growth_protocol: The human embryonic kidey 293T and murine brown preadipocyte line (iBA) cell lines were cultured to 90% confluency in Glutamax DMEM supplemented with FBS and penicillin-streptomycin.

extract_protocol:
Prior to use, cells were washed with PBS, dissociated via Trypsin-EDTA, washed with PBS, BSA and counted with Trypan blue live-dead stain using Countess cell counter (Invitrogen). Cells were mixed in a 1:1 ratio, adjusted to 20 cells/µl, and re-suspended in a cell buffer containing PBS, BSA, and Optiprep previous to loading onto the DisCo chip.
After bead-cell in droplet co-encapsulation, the gel loading tip containing the sample droplets was transferred to a bead collection chip inlet (cp-chip; as described in Lab on a chip). Droplets in the tip were flushed to bead collection chip. Subsequently to bead capture, washing was performed as in Drop-seq with SSC and Reverse transcription buffer directly in the recovery chip. Reverse transcription solution was added to the beads and the recovery chip was placed on a heating block to perform first strand cDNA synthesis (RT) for 90 minutes at 42oC. After RT reaction beads were washed on the recovery chip with TE-SDS once, with TE-TW twice and with Tris once. The beads were treated with Exonuclease I for 45 minutes at 37oC to remove single-stranded oligonucleotides on beads. After Exonuclease I treatment, beads were washed with TE-SDS once, with TE-TW twice (as after reverse transcription). Beads were then eluted from the recovery chip in dH2O. cDNA was amplified for 18 – 23 cycles using Kapa HiFi Hot start ready mix. cDNA was purified with CleanPCR magnetic beads using 0.6X ratio to remove small cDNA fragments and primers. To assess the cDNA yield and quality concentration was measured using Qubit and cDNA traces quality was assessed using Fragment Analyzer (Agilent). cDNA was tagmented with in-house Tn5 (generated as described in Picelli et al..) and the library was purified using CleanPCR magnetic beads (0.6X ratio).
Libraries were sequenced on a NextSeq 500 system (Illumina) following recommendations from original protocol (20 bp for read 1 and 50 bp for read2).

tag: culture method

value: In vitro cell culture

tag: passages

value: mixture of HEK 293T and iBA


title: D. melanogaster macrophages_DfosDN_Rep1

description: FACS-sorted labled macrophages


title: D. melanogaster macrophages_DfosDN_Rep2

description: FACS-sorted labled macrophages


title: ADT_EXP20001260_Sample_05_hashed sample

description: ADT