title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_2_P1-08C]

description: sci-RNA-seq3 library


title: CD68 DSP-1001660005232-A-H04

status: Public on Apr 12 2023

type: SRA

city: La Jolla

first: Judith

last: Varner

state: CA

address: 3855 Health Sciences Dr.

department: Moores Cancer Center

country: USA

institute: University of California, San Diego

description:
CD68
RNA (amplified oligo nucleotide probes)

library_source: transcriptomic

data_processing:
Counts from barcodes corresponding to RNA probes were normalized with internal positive and negative controls and then normalized to ROI area.
Data were pre-processed in nanoString DSP Software v 2.2.0.123 with Q3 normalization. ROIs were classified according to predominant cell type (epithelium, granulocyte, macrophage) in one of two conditions (COVID-19 positive and normal). The R BioConductor packages edgeR (Robinson et al., 2010) and limma (Ritchie et al., 2015) were used to implement the limma-voom (Law et al., 2014) method for differential expression analysis within and across cell types and condition. Trimmed mean of M-values (TMM) (Robinson and Oshlack, 2010) normalization was applied. The experimental design was modeled upon cell type and condition (~0 + cellType_condition). The voom method was employed to model the mean-variance relationship, after which lmFit was used to fit per-gene linear models and empirical Bayes moderation was applied with the eBayes function. Significance was defined by using an adjusted p-value cut-off of <0.05 after multiple testing correction (Benjamini and Hochberg, 1995) using a moderated t-statistic in limma.
Genome_build: GCA_000001405.15
Supplementary_files_format_and_content: Nanostring GeoMx Q3 normalized data file as csv file,  Included relative expression levels of genes

molecule: total RNA

organism: Homo sapiens

source_name: Normal Lung

extract_protocol:
nanoString GeoMx digital spatial profiling was applied to 3 COVID-19  and 1 normal lung tissues to acquire spatially resolved, quantitative measurements of gene expression in human lung macrophages, granulocytes and epithelium in FFPE tissue sections. Three cases of post-mortem COVID lung tissues and one case of surgically resected normal lung tissue were sectioned, deparaffinized, rehydrated and treated with low pH retrieval solution. nanoString GeoMX Digital Spatial ImmuneOncology and COVID-19 RNA probes were hybridized to sections overnight. Sections were then stained with a cocktail of immunofluorescent antibodies comprised of Alexa fluor 532-labeled anti-CD68 (Clone KP1, 1:100, Novus Biologicals, USA), Alexa fluor 647-labeled Myeloperoxidase (Clone 2C7, 1:500, Novus Biologicals, USA), Alexa fluor 594 labelled anti-PanCytokeratin (C AE-1, AE-3) and Alexa fluor 488 SYTO13 Nuclear stain.  Digital scanning of slides was performed on the GeoMX Digital Profiler (nanoString Technologies, Inc.). Regions of Interest (ROI) and cell type with a spatial resolution of approximately 10 mm were selected, exposed to UV light for RNA probe cleavage and oligonucleotide collection.
Samples were sequenced on a NovaSeq6000.

tag: geomx oligo set

value: Cancer Transcriptome Atlas, (v1.0) GeoMx COVID-19

tag: roi number

tag: ffpe

value: FFPE tissue


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_2_P1-07F]

city: Seattle

first: Xiaoyi

last: Li

state: WA

address: 3720 15th Ave NE

department: Department of Genome Sciences

institute: University of Washington

description: sci-RNA-seq3 library

data_processing:
For the sci-RNA-seq3 transcriptome libraries, sequencing reads from the Illumina NextSeq platforms were first demultiplexed based on PCR index using the bcl2fastq software. Reads were filtered based on RT and ligation index, by allowing <2 hamming distance from reference. Filtered reads were trimmed with TrimGalore (v0.6.6) with parameters: -a AAAAAAAA --three_prime_clip_R1 1. Reads were then aligned to the GRCh38 reference genome using STAR (v2.7.6a). PCR duplicates were collapsed using the RT index, ligation index, UMI sequence and end coordinate of reads. Reads were further demultiplexed based on the combination of the RT, ligation and PCR index and split into files for individual cells. To generate gene expression count matrices, reads were assigned to the exonic and intronic region of closest genes with HTseq (v.2.0.2). Reads with ambiguous assignments were discarded. Cells were further filtered based on total UMI (> 100) and mitochondrial reads percentage (<10%). Cells with the number of features detected between 10% and 90% percentile of all cells were kept and considered high-quality cells. The single cell analysis was performed using the Seurat (v4.0.0) package in R.
For capture libraries of the synHEK3 and shRNA constructs, sequencing reads from the Illumina NextSeq platforms were first demultiplexed based on PCR index using the bcl2fastq software. A custom script was used to further demultiplex and filter the synHEK3 and shRNA libraries based on the combination of RT, ligation and PCR index (cell ID), by allowing <2 hamming distance from reference. For the synHEK3 library, reporter barcodes (<2 hamming distance to the reference barcode set), prime editing outcomes and read UMIs were extracted. For the shRNA library, shRNA sequences (<2 hamming distance to the reference barcode set) and read UMIs were extracted. UMIs <3 hamming distance were collapsed.   A series of pre-processing steps were applied to the data. 1) We matched cell IDs between the sci-RNA-seq3 transcriptome libraries and the capture libraries, and only kept high-quality cells (number of features between 10% and 90% percentile). 2) For the shRNA library, cells with <3 or >200 UMIs or >20 shRNA were removed. 3) For the synHEK3 library, cells with <4 UMIs were first removed. We counted UMIs for synHEK3 reporters belonging to the two clones and calculated a clone UMI/total UMI ratio. If the ratio was >80% for a specific clone, the cell is assigned to the corresponding clone. Cells with extremely high UMIs (cutoff between 100 and 250 UMIs, depending on library complexity and sequencing depth of the plate) were also removed for downstream analysis. 4) For a cell-synHEK3 barcode combination, if multiple insertion sequences were observed, sequences <3 hamming distance were collapsed. Cell-synHEK3 barcode pairs having conflicting editing outcomes were discarded. 5) A cell-synHEK3 barcode-editing outcome-shRNA matrix was generated for a common set of cells identified by the shRNA and synHEK3 libraries.
Assembly: GRCh38
Supplementary files format and content: cell_synHEK3_shRNA.csv.gz contains a cell-synHEK3-shRNA capture matrix

molecule: polyA RNA

growth_protocol: K562 cells (CCL-243) were purchased from ATCC and maintained in RPMI 1640 medium (Gibco) supplied with 10% FBS (Hyclone) and penicillin/streptomycin (Gibco, 100 U/ mL).

extract_protocol: We performed a modified version of sci-RNA-seq3 (Martin et al., 2022). In brief, K562 cells were lysed with 5 mL of a hypotonic, PBS-based lysis buffer containing DEPC. The resulting nuclei were fixed with 4 volume of ice-cold methanol. After rehydrating and washing the nuclei carefully in a sucrose/PBS/triton buffer (SPBST), the nuclei were treated with T7 polymerase in an NTP mix buffer at 37oC for 30 min. Then nuclei were washed, sonicated briefly and counted. The nuclei were distributed to a 96-well plate for reverse transcription, with a cocktail of three indexed primers: oligo-dT, HEK3 and CS1 -- specific for the cellular transcriptome, and RNAs produced from the synHEK3 reporter and the shRNA constructs, respectively. After reverse transcription, nuclei were pooled, washed in SPBST and redistributed to 4 fresh plates for ligation of the second index primer with T4 DNA ligase. Nuclei were then again pooled, washed, and redistributed to 4 final plates at two different concentrations (1300 cell per well for 2 plates and 2500 cell per well for 2 plates) for second strand synthesis and extraction. Nuclear lysates were split in half, with one half undergoing normal sci-RNA-seq3 transcriptome library preparation (tagmentation and PCR to add the third index) and the other half subjected to indexed enrichment PCR for both the synHEK3 and shRNA constructs. Products were pooled by PCR plate, size-selected and sequenced on an Illumina Nextseq 2000 or Nextseq 550.

treatment_protocol: Two monoclonal K562 PE2-Puro lines (clone 3 and clone 5) containing multiple randomly integrated chromatin sensors for prime editing (synHEK3) were transduced with Lenti-rtTA-P2A-Blast viruses and selected in 10 ug/mL Blasticidin for 7 days. Monoclonal lines were derived and further transduced with a DNA damage response gene-focused lentiviral shRNA library containing 304 hairpins targeting 76 genes. Cells were transdcued at either low MOI (<1) or high MOI (~10) and selected in 800 ug/mL Geneticin for 7 days. For preparing sci-RNA-seq3 libraries, at the RT step, the low MOI cells are placed in the first 6 columns of the 96-well plate, while the high MOI cells were placed in the 7-12 columns of the 96-well palte. Low MOI and High MOI transduced cells can be distiguished by RT indices.

tag: tissue

tag: cell line

tag: cell type

value: lymphoblast

tag: treatment

value: lentiviral transduction of a DDR-focused shRNA library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_2_P1-07E]

description: sci-RNA-seq3 library


title: PanCK DSP-1001660005232-A-H02

description:
PanCK
RNA (amplified oligo nucleotide probes)


title: CD68 DSP-1001660005232-A-H01


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_2_P1-07C]

description: sci-RNA-seq3 library


title: Myeloperoxidase DSP-1001660005232-A-H03

description:
Myeloperoxidase
RNA (amplified oligo nucleotide probes)


title: Myeloperoxidase DSP-1001660005232-A-G12


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_2_P1-07B]

description: sci-RNA-seq3 library


title: PanCK DSP-1001660005232-A-G11


title: NB1

description:
TempoSeq lysate containing RNA
Human normal lung tissue  derived from surgical specimens acquired during lung cancer surgeries and COVID19 postmortem tissues were fixed 48h in Z-fix then embedded in paraffin and 5 micron sections were prepared.

data_processing:
Unnormalized read counts were obtained from BioSpyder TempO-seq. The R BioConductor packages edgeR (Robinson et al., 2010) and limma (Ritchie et al., 2015) were used to implement the limma-voom (Law et al., 2014) method for differential expression analysis. We analyzed normal lung, COVID-19 lung and COVID-19 BALF sample groups together and also lung samples alone due to low levels of alignment in the BALF samples. We also analyzed COVID-19 BALF and normal BALF together. One COVID-19 BALF sample was removed from downstream analysis because it had <300,000 quantified reads. Lowly expressed genes—those not having counts per million (cpm)  10 in at least 10 of the samples—were filtered out in the combined analysis and for lung samples only a less stringent filter of (cpm)  1 in at least 5 of the samples was applied. Trimmed mean of M-values (TMM) normalization (Robinson and Oshlack, 2010) was applied after filtering. The experimental design was modeled upon condition, called condition (~0 + condition). The voom method was employed to model the mean-variance relationship, after which lmFit was used to fit per-gene linear models and empirical Bayes moderation was applied with the eBayes function. Significance was defined by using an adjusted p-value cut-off of <0.05 after multiple testing correction (Benjamini and Hochberg, 1995) using a moderated t-statistic in limma. Functional enrichment of the differentially expressed genes was performed using SPIA (Tarca et al., 2009), gProfiler (Raudvere et al., 2019) and fGSEA (Korotkevich et al., 2021).
Genome_build: GCA_000001405.15
Supplementary_files_format_and_content: Csv file with raw gene counts for every gene and every sample

source_name: FFPE Normal human brochoalveolar lavage cells

extract_protocol:
Two five-micron FFPE sections from each of twelve postmortem lung specimens from COVID-19 patients, five normal lung specimens, eight BALF specimens from COVID-19 patients and five BALF specimens from normal patients were used to perform TempoSeq (Templated Oligo assay with Sequencing readout) FFPE human whole transcriptome RNA sequencing at BioSpyder Technologies, Inc, Carlsbad, CA, as described (Trejo et al., 2019; Turnbull et al., 2020). In short, two 5 FFPE tissue sections per sample were scraped from glass slides, paraffin removed and at least two 25 nucleotide long oligonucleotides specific for 19,283 genes (21,111 probes) were used to prepare full length (50 nucleotide-long) probes that were amplified prior to sequencing library preparation. Prepared libraries were sequenced on NovaSeq6000; mapped reads were generated by TempO-SeqR alignment of demultiplexed FASTQ files using Bowtie, allowing for up to 2 mismatches in the 50-nucleotide target sequence.
Prepared libraries were sequenced on HiSeq 2500 using the non-patterned flow cells to avoid index hopping

value: bronchoalveolar lavage

tag: tissue processing

value: FFPE and sectioned cell pellets

tag: normal vs covid-19

value: Normal

tag: tissue from postmortem or living patient

value: Living


title: Myeloperoxidase DSP-1001660005232-A-H06


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_2_P1-06H]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_2_P1-12C]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_2_P1-07A]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_2_P1-07G]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_2_P1-06G]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_2_P1-06F]

description: sci-RNA-seq3 library


title: CD68 DSP-1001660005232-A-G10


title: PanCK DSP-1001660005232-A-G08


title: Myeloperoxidase DSP-1001660005232-A-G09


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_2_P1-06E]

description: sci-RNA-seq3 library


title: CD68 DSP-1001660005232-A-G07


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_2_P1-06D]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_2_P1-06C]

description: sci-RNA-seq3 library


title: Myeloperoxidase DSP-1001660005232-A-G06


title: CD68 DSP-1001660005232-A-G04


title: PanCK DSP-1001660005232-A-G05


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_2_P1-07D]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_2_P1-06B]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_2_P1-06A]

description: sci-RNA-seq3 library


title: Myeloperoxidase DSP-1001660005232-A-G03


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_2_P1-05H]

description: sci-RNA-seq3 library


title: PanCK DSP-1001660005232-A-G02


title: CD68 DSP-1001660005232-A-G01


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_2_P1-05G]

description: sci-RNA-seq3 library


title: Myeloperoxidase DSP-1001660005232-A-F12


title: PanCK DSP-1001660005232-A-F11


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_2_P1-05F]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_2_P1-05E]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_2_P1-05D]

description: sci-RNA-seq3 library


title: CD68 DSP-1001660005232-A-F10


title: Myeloperoxidase DSP-1001660005232-A-F09


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_2_P1-05C]

description: sci-RNA-seq3 library


title: PanCK DSP-1001660005232-A-F08


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_2_P1-04H]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_2_P1-05A]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_2_P1-05B]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_2_P1-04G]

description: sci-RNA-seq3 library


title: Myelop[eroxidase DSP-1001660005232-A-F06

description:
Myelop[eroxidase
RNA (amplified oligo nucleotide probes)

source_name: C13 COVID LUNG


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_2_P1-04E]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_2_P1-04F]

description: sci-RNA-seq3 library


title: Myelop[eroxidase DSP-1001660005232-A-F03


title: PanCK DSP-1001660005232-A-F02


title: CD68 DSP-1001660005232-A-F01


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_2_P1-04D]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_2_P1-04C]

description: sci-RNA-seq3 library


title: PanCK DSP-1001660005232-A-E11


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_2_P1-04B]

description: sci-RNA-seq3 library


title: CD68 DSP-1001660005232-A-E10


title: Myelop[eroxidase DSP-1001660005232-A-E12


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_2_P1-04A]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_2_P1-03H]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_2_P1-03G]

description: sci-RNA-seq3 library


title: CD68 DSP-1001660005232-A-F07


title: Myelop[eroxidase DSP-1001660005232-A-E09


title: PanCK DSP-1001660005232-A-E08


title: CD68 DSP-1001660005232-A-E07


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_2_P1-03E]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_2_P1-03F]

description: sci-RNA-seq3 library


title: Myelop[eroxidase DSP-1001660005232-A-E06


title: PanCK DSP-1001660005232-A-F05


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_2_P1-03D]

description: sci-RNA-seq3 library


title: CD68 DSP-1001660005232-A-F04


title: PanCK DSP-1001660005232-A-E05


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_2_P1-03C]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_2_P1-03B]

description: sci-RNA-seq3 library


title: PanCK DSP-1001660005232-A-E02


title: Myelop[eroxidase DSP-1001660005232-A-E03


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_2_P1-03A]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_2_P1-02G]

description: sci-RNA-seq3 library


title: CD68 DSP-1001660005232-A-E01


title: Myelop[eroxidase DSP-1001660005232-A-D12


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_2_P1-02E]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_2_P1-02F]

description: sci-RNA-seq3 library


title: CD68 DSP-1001660005232-A-D10


title: Myelop[eroxidase DSP-1001660005232-A-D09


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_2_P1-02D]

description: sci-RNA-seq3 library


title: PanCK DSP-1001660005232-A-D08


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_2_P1-02B]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_2_P1-02A]

description: sci-RNA-seq3 library


title: CD68 DSP-1001660005232-A-D07


title: CD68 DSP-1001660005232-A-E04


title: PanCK DSP-1001660005232-A-D04

source_name: C11 COVID LUNG


title: Myeloperoxidase DSP-1001660005232-A-D05


title: CD68 DSP-1001660005232-A-D03


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_2_P1-01G]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_2_P1-01E]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_2_P1-01F]

description: sci-RNA-seq3 library


title: CD68 DSP-1001660005232-A-D06


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_2_P1-02H]

description: sci-RNA-seq3 library


title: Myeloperoxidase DSP-1001660005232-A-D02


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_2_P1-01D]

description: sci-RNA-seq3 library


title: PanCK DSP-1001660005232-A-D01


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_2_P1-01C]

description: sci-RNA-seq3 library


title: CD68 DSP-1001660005232-A-C12


title: PanCK DSP-1001660005232-A-D11


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_2_P1-01B]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_2_P1-02C]

description: sci-RNA-seq3 library


title: Myeloperoxidase DSP-1001660005232-A-C11


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_2_P1-01A]

description: sci-RNA-seq3 library


title: Myeloperoxidase DSP-1001660005232-A-C09


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_P1-12G]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_2_P1-01H]

description: sci-RNA-seq3 library


title: PanCK DSP-1001660005232-A-C08


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_P1-12E]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_P1-12D]

description: sci-RNA-seq3 library


title: CD68 DSP-1001660005232-A-C07


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_P1-12C]

description: sci-RNA-seq3 library


title: CD68 DSP-1001660005232-A-C05


title: Myeloperoxidase DSP-1001660005232-A-C04


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_P1-12B]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_2_P1-09G]

description: sci-RNA-seq3 library


title: Myeloperoxidase DSP-1001660005232-A-C06


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_P1-12A]

description: sci-RNA-seq3 library


title: aco-mutant-BA

city: Cincinnati

first: Nathan

last: Salomonis

state: OH

address: 3333 Burnet Avenue

department: Biomedical Informatics

institute: Cincinnati Children's Hospital

data_processing:
The scRNA-Seq FASTQ files (10x Genomics 3' version 3) were aligned to the Zebrafish Ensembl version 91 reference genome and transcriptome (GRCz11), using Cell Ranger version 3.1.0. Cell Ranger filtered feature sparse matrix counts files from all samples (HDF5 format) were supplied to AltAnalyze version 2.1.4 for unsupervised analysis using the EnsMart 91 zebrafish database and ICGS2 algorithm with default parameters. Cell-type predictions were initially obtained from ICGS2 based on marker enrichment and then further refined based on manual curation with literature-associated markers. The obtained ICGS2 clusters were filtered to include muscle, endothelial and epithelial clusters for downstream UMAP visualization.
Genome_build: hg19
Supplementary_files_format_and_content: HDF5 sparse matrices and Excel workbook

organism: Danio rerio

source_name: Heart

extract_protocol:
The SV and BA were dissected from using fine forceps and pooled in a Tyrode’s solution (pH 7.4) following washing in 1X PBS. The pooled tissue was washed 3 times in Tyrode’s before the addition of Liberase IV (0.29U/ml, Roche, 05401020001), Elastase (1.0 U/ml, Sigma, E7885), and Pronase E (0.92 U/ml, Sigma, P5147). Dissociation of the tissues was facilitated via incubation at 37°C and gentle trituration with pipettes of descending tip diameter (first 2.0 mm followed by 0.8mm. Can also use filtered P1000 and P20 tips, respectively). The dissociation reaction was halted by transferring the cells to a modified Kraft-Brühe solution (pH 6.9) and the cells were spun down to a pellet before being resuspended in a final 1% BSA solution in 1X PBS.
The 10x Genomics Chromium 3’ v2 assay standard protocol was followed as previously described.
Sequencing was performed on the Illumina HiSeq-4000 platform at a target depth of 230 million reads per simple.

tag: age

value: Adult

value: Bulbous Arteriosus

tag: genotype

value: nr2f1a mutant


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_P1-11H]

description: sci-RNA-seq3 library


title: CD68 DSP-1001660005232-A-C10


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_P1-11G]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_P1-12H]

description: sci-RNA-seq3 library


title: aco-mutant-SV

value: Sinus Venosus


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_P1-11E]

description: sci-RNA-seq3 library


title: CD68 DSP-1001660005232-A-C03


title: BA-WT-Control

value: WT


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_P1-11F]

description: sci-RNA-seq3 library


title: Myeloperoxidase DSP-1001660005232-A-C02


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_P1-11D]

description: sci-RNA-seq3 library


title: CD68 DSP-1001660005232-A-C01


title: PanCK DSP-1001660005232-A-B11

source_name: C8 COVID LUNG


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_P1-12F]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_P1-11B]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_P1-11C]

description: sci-RNA-seq3 library


title: SV-WT-Control


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_P1-11A]

description: sci-RNA-seq3 library


title: Achilles tendon, pefloxacin and dexamethasone [PD3]

city: Kyoto

first: Kazuki

last: Nagayasu

address: 46-29 Yoshidashimoadachicho, Sakyo

department: Graduate School of Pharmaceutical Sciences

country: Japan

institute: Kyoto University

data_processing:
The reads were cleaned using SOAPnuke v1.5.2
The clean reads were mapped to the genome and gene sequences using HISAT and Bowtie2, respectively.
Gene expression was calculated using RSEM.
Assembly: GCF_000001895.5_Rnor_6.0

organism: Rattus norvegicus

source_name: Achilles tendon

extract_protocol:
After chronic treatment, Achilles tendons were collected and snap frozen in liquid N2. Total RNA wa isolated using ISOGEN reagent.
RNA libraries were prepared for sequencing using standard BGISEQ protocols

value: Achilles tendon

tag: strain

value: Wistar/ST

value: Pefloxacin for 4 weeks followed by 1 week withdrawal, and dexamethasone for 5 weeks


title: Myeloperoxidase DSP-1001660005232-A-B10


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_P1-10G]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_P1-10E]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_P1-10F]

description: sci-RNA-seq3 library


title: Achilles tendon, dexamethasone [D2]

value: Dexamethasone for 5 weeks


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_P1-10D]

description: sci-RNA-seq3 library


title: PanCK DSP-1001660005232-A-B09

value: Cancer Transcriptome Atlas, (v1.0) GeoMx COVID-19


title: CD68 DSP-1001660005232-A-B08

value: FFPE tissue


title: Achilles tendon, pefloxacin [P2]

value: Pefloxacin for 4 weeks and withdrawal for 1week


title: Achilles tendon, vehicle [C2]

value: Vehicle for 5 weeks


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_P1-10C]

description: sci-RNA-seq3 library

value: lymphoblast

value: lentiviral transduction of a DDR-focused shRNA library


title: Myeloperoxidase DSP-1001660005232-A-B07


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_P1-10A]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_P1-10B]

description: sci-RNA-seq3 library


title: PanCK DSP-1001660005232-A-B06


title: 16N

city: Philadelphia

first: Stephanie

last: Daws

state: PA

address: 3500 N Broad St

institute: Temple University

data_processing:
Basecalling was performed with BGI proprietary software
Raw sequencing reads were filtered to remove reads with low quality tags, 5’ primer contaminants, tags without a 3’ primer, tags with poly A and those shorter than 18nt and the remaining reads were stored in FASTQ format.
Bowtie2 was used to align clean reads to the reference genome (rn6).
miRDeep2 was used to predict novel miRNA based on harpin structure of miRNA precursors.
miRNAs expression level was calculated as Transcripts Per Kilobase Million (TPM) to directly compare differences in gene expression between samples.
Assembly: rn6
Supplementary files format and content: raw counts

source_name: orbitofrontal cortex

extract_protocol:
Total RNA was isolated using the miRvana PARIS kit (AM1556) from Invitrogen according to the manufacturer's protocol.
Small-RNA sequencing was performed on four biological replicates per group by BGI Genomics (BGI Americas Corp, Cambridge, MA, USA). Total RNA was assessed for quality before library preparation and RNA Integrity Numbers (RIN) were ≥ 7.5 with 28S/18S ≥ 1.3. Small RNAs (18-30 nt) were separated from total RNA by PAGE gel. Selected small RNAs were ligated with 3’ and 5’ adapters. The formed strands were reverse transcribed in cDNA and PCR amplified with high-ping polymerase. The PCR products (100-120bp) were separated by PAGE gel to eliminate primer-dimers and other byproducts and purified. The PCR yield was quantified followed by single strand DNA cyclization (ssDNA circle) for final library construction. DNA nanoballs (DNBs) were generated by ssDNA circle by rolling circle replication. The DNBs were then loaded onto the BGISEQ-500 platform sequencing for 100bp paired end reads.

treatment_protocol: Adult male Sprague Dawley rats were aged 8 weeks on arrival. Forheroin exposure, animals were acclimited to the animal facility for 1 week then underwent jugular vein catheterization. Following 1 week of recovery, animals self-administered  heroin at a low dosage (HL-0.03mg/kg/infusion) or a high dosage (HH- 0.075mg/kg/infusion) for 6 hours per day in 10 daily sessions. Naive animals were briefly handled each day. Following the last self-administration session, animals remained in their homecage for 21 days. After 21 days, animals were euthanized and their brains were frozen in ice cold isopentane. Orbitofrontal cortex tissue was dissected from each animal on dry ice then RNA extraction proceeded.

value: Sprague Dawley

tag: Sex

value: male

value: adult

value: naïve, handled

value: orbitofrontal cortex


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_P1-09F]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_P1-09H]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_P1-09G]

description: sci-RNA-seq3 library


title: Myeloperoxidase DSP-1001660005232-A-B04


title: 14N


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_P1-10H]

description: sci-RNA-seq3 library


title: 13N


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_P1-09C]

description: sci-RNA-seq3 library


title: 8N


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_P1-09B]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_P1-09A]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_P1-08H]

description: sci-RNA-seq3 library


title: Myeloperoxidase DSP-1001660005232-A-B01


title: PanCK DSP-1001660005232-A-B03


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_P1-08G]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_P1-08F]

description: sci-RNA-seq3 library


title: 6N


title: PanCK DSP-1001660005232-A-A12


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_P1-09E]

description: sci-RNA-seq3 library


title: 71H

value: 10 days of heroin self-administration (0.03mg/kg)


title: CD68 DSP-1001660005232-A-A11


title: CD68 DSP-1001660005232-A-B05


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_P1-08E]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_P1-08C]

description: sci-RNA-seq3 library


title: 78H


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_P1-08A]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_P1-08D]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_P1-09D]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_P1-07H]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_P1-08B]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_P1-07G]

description: sci-RNA-seq3 library


title: CD68 DSP-1001660005232-A-B02


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_P1-07F]

description: sci-RNA-seq3 library


title: 15N


title: PanCK DSP-1001660005232-A-A09


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_P1-07E]

description: sci-RNA-seq3 library


title: 5N


title: 73H


title: Myeloperoxidase DSP-1001660005232-A-A07


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_P1-07C]

description: sci-RNA-seq3 library


title: 124H

value: 10 days of heroin self-administration (0.075mg/kg)


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_P1-07B]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_P1-07D]

description: sci-RNA-seq3 library


title: 122H


title: PanCK DSP-1001660005232-A-A06


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_P1-06H]

description: sci-RNA-seq3 library


title: Myeloperoxidase DSP-1001660005232-A-A10


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_P1-07A]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_P1-06G]

description: sci-RNA-seq3 library


title: 115H


title: 113H


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_P1-06E]

description: sci-RNA-seq3 library


title: Myeloperoxidase DSP-1001660005232-A-A04


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_P1-06F]

description: sci-RNA-seq3 library


title: 72H


title: PanCK DSP-1001660005232-A-A03


title: CD68 DSP-1001660005232-A-A08


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_P1-06D]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_P1-06C]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_P1-06A]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_P1-06B]

description: sci-RNA-seq3 library


title: CD68 DSP-1001660005232-A-A02


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_P1-05H]

description: sci-RNA-seq3 library


title: Strep. uberis1

type: RNA

city: Rehovot

first: Nahum

middle: Yehuda

last: Shpigel

state: Please Select…

address: POB 12

department: Veterinary

country: Israel

institute: Hebrew University

hyb_protocol: Hybridization was performed according to manufacturer's instructions.

scan_protocol: nCounter Sprint profiler was used for scanning and image acquisition.

data_processing: Data was analyzed using nSolver™ analysis software version 4.0

organism: Mus musculus

source_name: Mammary gland, Streptococcus uberis challenged

label_protocol: Labeling was performed according to manufacturer's instructions.

extract_protocol: Total RNA was extracted using the GeneElute Mammalian Total RNA Miniprep Kit (Sigma-Aldrich, Rehovot, Israel) combined with on-Column DNase I Digestion Set (Sigma) and stored at -80C.

treatment_protocol: Mice were infected with E. coli, Strep. uberis or M. bovis bacteria by intramammary infusion. After 24 or 48 hours, mammary tissues were sampled for RNA extraction.

value: mammary gland

value: Streptococcus uberis challenged


title: CD68 DSP-1001660005232-A-A05


title: Strep. uberis3


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_P1-05E]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_P1-05D]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_P1-05C]

description: sci-RNA-seq3 library


title: Mycoplasma bovis PG45-3

source_name: Mammary gland, Mycoplama bovis challenged

value: Mycoplama bovis challenged


title: Mycoplasma bovis PG45-2


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_P1-05B]

description: sci-RNA-seq3 library


title: RPS3+/- input, replicate 2

first: Tal

last: Havkin-Solomon

address: 234 Herzl

department: Biomolecular Sciences

institute: Weizmann Institute of Science

description:
RPS3+/- input, replicate 2
II2

data_processing:
Polysome profile reads were aligned to the human reference genome (hg38) using Bowtie2
The number of reads per gene was counted from the uniquely aligned reads in genecode (GRCh38.103) genomic regions using featureCounts
Normalization between samples was performed using the DESeq2 package in the R statistical programming language
Replicates similarities were tested using principal component analysis (PCA) and Pearson correlations and plotted using ggplot2 in R
The normalized gene read counts table (DESeq2 output table) was filtered to include only genes in high expression: only genes that the sample with the least number of reads per gene has > 32 reads were used for downstream analysis.
The normalized counts were used to calculate translation efficiency (TE)
The normalized counts were used to calculate translation efficiency (TE) (See manuscript: Havkin-Solomon et al.)
Assembly: hg38
Supplementary files format and content: Content: Gene reads coverage. Based on human gencode genes. Result of featureCount (txt, tabular format). Non-normalized.
Supplementary files format and content: File name: RPS3_project_Feb2021.gene_expression_levels.protein_coding_genes.with_gene_symbol_and_biotype.diff_lanes_sum_togther.RPS3_project.txt

source_name: kidney

growth_protocol: cells were grown at 37oC in a humidified incubator with 5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM; GIBCO, Life Technologies, Thermo Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (GIBCO), 100 units/mL penicillin and 100 ug/mL streptomycin.

extract_protocol:
cells were washed twice with cold buffer containing 20 mM Tris pH 8, 140 mM KCl, 5 mM MgCl2 and 100 µg/ml CHX. The cells were collected and lysed with 500 µl of the same buffer containing 0.5% Triton, 0.5% DOC, 1.5 mM DTT, 150 units RNAse inhibitor (Eurx) and 5 µl of protease inhibitor (Sigma). The lysed samples were centrifuged at 12,000g at 4˚C for 5 min. The cleared lysates were loaded onto a 10–50% sucrose gradient and centrifuged at 38,000 RPM in an SW41 rotor for 105 min at 4˚C. Gradients were fractionated and the optical density at 254 nm was continuously recorded using the ISCO absorbance detector UA-6. The collected samples were merged to create the High translated fractions (3+ ribosomes). The RNA of these fractions and the Input samples were isolated using Trizol and Direc-Zol RNA mini-prep kits (Zymo Research).   For global translation analysis, the collected samples were merged to create the High translated fractions (3+ ribosomes). The RNA of these fractions and the Input samples were isolated using Trizol and Direc-Zol RNA mini-prep kits (Zymo Research).
Equivalent RNA concentrations were taken from each fraction for the library preparation for MARS-Seq

treatment_protocol: cells were incubated with 100 µg/ml Cycloheximide (CHX, Sigma) for 5 min

value: kidney

value: HEK293 TAF1 ts mutant cells

value: un-treated


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_P1-05A]

description: sci-RNA-seq3 library


title: Mycoplasma bovis PG45-1


title: RPS3+/- high translated, replicate 1

description:
RPS3+/- high translated, replicate 1
IH


title: Strep. uberis2


title: RPS3+/- high translated, replicate 2

description:
RPS3+/- high translated, replicate 2
IH2


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_P1-05G]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_P1-04H]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_P1-05F]

description: sci-RNA-seq3 library


title: K148D input, replicate 1

description:
K148D input, replicate 1
148I


title: E. coli P4-NR3

source_name: Mammary gland, E. coli challenged

value: E. coli challenged


title: E. coli P4-NR1


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_P1-04G]

description: sci-RNA-seq3 library


title: 24_13633RRMS209.CEL

city: Ramat Gan

first: Michael

last: Gurevich

address: Tel Hashomer

department: Multiple Sclerosis Center

institute: Sheba Medical Center

hyb_protocol: Following fragmentation, 10 ug of cRNA were hybridized for 16 hr at 45C on GeneChip HU-133A-2. GeneChips were washed and stained in the Affymetrix Fluidics Station 400.

scan_protocol: GeneChips were scanned using the Hewlett-Packard GeneArray Scanner G2500A.

data_processing:
The raw CEL files were adjusted for GC content.
Probe sets summarized by median polish algorithm implemented in Partek software (www.partel.com)

label: biotin

source_name: PBMC

label_protocol: Biotinylated cRNA were prepared according to the standard Affymetrix protocol from 6 ug total RNA (Expression Analysis Technical Manual, 2001, Affymetrix).

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

treatment_protocol: PBMC were purified using Ficoll hypaque gradient (Invitrogen, USA).

tag: patient diagnosis

value: multiple sclerosis

value: PBMC

tag: age(years)

tag: gender

value: female

tag: batch number


title: RPS3+/- input, replicate 1

description:
RPS3+/- input, replicate 1
II


title: 24_13714NMS209.CEL


title: 24_13666NMS209.CEL


title: K148D input, replicate 2

description:
K148D input, replicate 2
148I2


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_P1-04F]

description: sci-RNA-seq3 library

value: lymphoblast

value: lentiviral transduction of a DDR-focused shRNA library


title: Normal3

source_name: Mammary gland, non-challenged

value: control, non-challenged


title: 24_13627NMS209.CEL


title: 24_13655NMS209.CEL

value: male


title: E. coli P4-NR2


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_P1-04E]

description: sci-RNA-seq3 library


title: 24_13614NMS209.CEL


title: Normal2


title: K148D high translated, replicate 2

description:
K148D high translated, replicate 2
148H2


title: K148D high translated, replicate 1

description:
K148D high translated, replicate 1
148H


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_P1-04D]

description: sci-RNA-seq3 library


title: Normal1


title: R146D input, replicate 1

description:
R146D input, replicate 1
146I


title: 24_13060NMS209.CEL


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_P1-04C]

description: sci-RNA-seq3 library


title: SjS10_S2_PBMC_control

city: Rotterdam

first: Pieter Martijn

last: Kolijn

address: Doctor Molewaterplein 40

department: Immunology

country: Netherlands

institute: Erasmus MC

description: IGH gene repertoire sequencing from pre-diagnostic SjS eMZL patients and controls

library_source: genomic

data_processing:
Raw FASTQ files were uploaded to the ARResT/Interrogate immunoprofiler for annotation and initial exploratory analysis. The full productive IGH repertoire for each sample was downloaded in .csv format and processed in R (R Core Team, 2020) during further analysis.
Assembly: GRCh38
Supplementary files format and content: .xlsx , quantitative processed data (abundance measurements)
Library strategy: DNA-seq

molecule: genomic DNA

source_name: Buffy coat

growth_protocol: Blood  samples were collected at from SjS patients, buffycoats and serum were stored at -80 °C until further analysis

extract_protocol:
Total  gDNA was isolated from the buffycoat DNA and 250-500 ng was amplified through multiplex PCR of IGHV-IGHD-IGHJ gene rearrangements. After amplification, PCR products were purified on Agencourt AMPURE beads (Beckman Coulter, Brea, CA, USA) and eluted using H2O. The purified product was checked for primer dimers using the Agilent Tapestation (Agilent,Santa Clara, CA, USA).
The intitial multiplex PCR already attached the indexes to the PCR products, so library construction was simply pooling the purified PCR product in equilmolar fashion.

value: leukocytes

tag: group

value: SjS control

tag: sample type

value: Control


title: 24_12032RRMS209.CEL

tag: tissue


title: SjS11_S1_PBMC_control


title: R146D input, replicate 2

description:
R146D input, replicate 2
146I2

tag: cell line


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_P1-04B]

description: sci-RNA-seq3 library

tag: cell type


title: SjS10_S1_PBMC_control


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_P1-04A]

description: sci-RNA-seq3 library

tag: treatment


title: SjS9_S2_PBMC_control


title: R146D high translated, replicate 2

description:
R146D high translated, replicate 2
146H2


title: R146D high translated, replicate 1

description:
R146D high translated, replicate 1
146H


title: 23_8661RRMS208.CEL


title: SjS9_S1_PBMC_control


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_P1-03H]

description: sci-RNA-seq3 library


title: R116D input, replicate 1

description:
R116D input, replicate 1
116I


title: SjS8_tissue_biopsy_diagnosis

source_name: Lung

value: Lung

value: SjS eMZL

value: diagnostic


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_P1-03G]

description: sci-RNA-seq3 library


title: R116D input, replicate 2

description:
R116D input, replicate 2
116I2

value: kidney


title: 23_13519NMS208.CEL


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_P1-03F]

description: sci-RNA-seq3 library


title: 23_13512NMS208.CEL


title: SjS7_tissue_biopsy_diagnosis

source_name: Lymph node

value: Lymph node


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_P1-03E]

description: sci-RNA-seq3 library


title: 23_13505NMS208.CEL


title: 23_7351NMS208.CEL


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_P1-03D]

description: sci-RNA-seq3 library


title: RPS26dC, Light fraction, replicate 1

description:
RPS26dC, Light fraction, replicate 1
D5L

data_processing:
Polysome profile reads were aligned to the human reference genome (hg38) using Bowtie2
The number of reads per gene was counted from the uniquely aligned reads in genecode (GRCh38.103) genomic regions using featureCounts
Normalization between samples was performed using the DESeq2 package in the R statistical programming language
Replicates similarities were tested using principal component analysis (PCA) and Pearson correlations and plotted using ggplot2 in R
The normalized gene read counts table (DESeq2 output table) was filtered to include only genes in high expression: only genes that the sample with the least number of reads per gene has > 32 reads were used for downstream analysis.
The normalized counts were used to calculate translation efficiency (TE)
The normalized counts were used to calculate translation efficiency (TE) (See manuscript: Havkin-Solomon et al.)
Assembly: hg38
Supplementary files format and content: Content: Gene reads coverage. Based on human gencode genes. Result of featureCount (txt, tabular format). Non-normalized.
Supplementary files format and content: File name: RPS26_project_Feb2021.gene_expression_levels.protein_coding_genes.with_gene_symbol_and_biotype.diff_lanes_sum_togther.RPS26_project.txt

extract_protocol:
Cells were washed twice with cold buffer containing 20 mM Tris pH 8, 140 mM KCl, 5 mM MgCl2 and 100 µg/ml CHX. The cells were collected and lyzed with 500 µl of the same buffer containing 0.5% Triton, 0.5% DOC, 1.5 mM DTT, 150 units RNAse inhibitor (Eurx) and 5 µl of protease inhibitor (Sigma). The lyzed samples were centrifuged at 12,000g at 4˚C for 5 min. The cleared lysates were loaded onto a 10–50% sucrose gradient and centrifuged at 38,000 RPM in an SW41 rotor for 105 min at 4˚C. Gradients were fractionated and the optical density at 254 nm was continuously recorded using the ISCO absorbance detector UA-6. The collected samples were merged to create the fractions: Light polysome (2-5 ribosomes) and Heavy (6+ ribosomes). The RNA of these fractions and the Input samples were isolated using Trizol and Direc-Zol RNA mini-prep kits (Zymo Research).
Equivalent RNA concentrations were taken from each fraction for the library preparation for MARS-Seq

treatment_protocol: Untreated and 4 hours glucose starved [glucose-free media (11966025, Gibco) with 10% dialyzed FBS] WT and RPS26dC cells were incubated with 100 µg/ml Cycloheximide (CHX, Sigma) for 5 min

value: HEK293 TAF1 ts mutant cells

value: un-treated


title: R116D high translated, replicate 2

description:
R116D high translated, replicate 2
116H2


title: 23_13503NMS208.CEL

value: multiple sclerosis


title: R116D high translated, replicate 1

description:
R116D high translated, replicate 1
116H


title: SjS7_PBMC_relapse

value: Pre eMZL diagnosis


title: 23_13429NMS208.CEL

value: PBMC

value: female


title: 23_13324PDMS213.CEL


title: SjS6_tissue_biopsy_diagnosis


title: 23_13295NMS208.CEL


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_P1-03C]

description: sci-RNA-seq3 library

value: lymphoblast

value: lentiviral transduction of a DDR-focused shRNA library


title: RPS26dC, Light fraction, replicate 2

description:
RPS26dC, Light fraction, replicate 2
D5L2


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_P1-03B]

description: sci-RNA-seq3 library


title: 23_10954RRMS208.CEL


title: SjS6_S4_PBMC


title: SjS6_S2_PBMC


title: SjS8_PBMC_diagnosis


title: RPS26dC, Input fraction, replicate 1

description:
RPS26dC, Input fraction, replicate 1
D5I


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_P1-03A]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_P1-02H]

description: sci-RNA-seq3 library


title: RPS26dC, Heavy fraction, replicate 1

description:
RPS26dC, Heavy fraction, replicate 1
D5H


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_P1-02G]

description: sci-RNA-seq3 library


title: 22_7741NMS207.CEL


title: RPS26dC, Heavy fraction, replicate 2

description:
RPS26dC, Heavy fraction, replicate 2
D5H2


title: SjS6_S1_PBMC


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_P1-02F]

description: sci-RNA-seq3 library


description:
RPS26dC, Glucose starvation, Light fraction, replicate 1
D0L

value: Glucose starvation


title: RPS26dC, Input fraction, replicate 2

description:
RPS26dC, Input fraction, replicate 2
D5I2


title: SjS5_tissue_biopsy_diagnosis

source_name: Parotid gland

value: Parotid gland


title: 22_9259RRMS207.CEL


description:
RPS26dC, Glucose starvation, Light fraction, replicate 2
D0L2


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_P1-02E]

description: sci-RNA-seq3 library


title: SjS5_S1_PBMC

value: leukocytes


title: 23_12888RRMS208.CEL


title: 22_13455NMS207.CEL

value: male


title: SjS4_PBMC


title: SjS6_S3_PBMC


description:
RPS26dC, Glucose starvation, Input fraction, replicate 1
D0I


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_P1-02C]

description: sci-RNA-seq3 library


title: 22_10988NMS207.CEL


title: 22_13055NMS207.CEL


description:
RPS26dC, Glucose starvation, Input fraction, replicate 2
D0I2


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_P1-02B]

description: sci-RNA-seq3 library


title: 22_13294NMS207.CEL


title: SjS3_S5_tissue_biopsy

source_name: Breast

value: Breast


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_P1-02A]

description: sci-RNA-seq3 library


title: SjS3_S4_tissue_biopsy

source_name: Bronchial

value: Bronchial

value: SjS eMZL


description:
RPS26dC, Glucose starvation, Heavy fraction, replicate 1
D0H

value: kidney


description:
RPS26dC, Glucose starvation, Heavy fraction, replicate 2
D0H2


title: SjS3_S3_tissue_biopsy

value: Lymph node


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_P1-02D]

description: sci-RNA-seq3 library


title: SjS3_S2_tissue_biopsy


title: 22_13038RRMS207.CEL


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_P1-01H]

description: sci-RNA-seq3 library


title: 22_10711RRMS207.CEL


title: WT, Light fraction, replicate 2

description:
WT, Light fraction, replicate 2
2935L2

value: HEK293 TAF1 ts mutant cells

value: WT


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_P1-01G]

description: sci-RNA-seq3 library


title: SjS3_S1_tissue_biopsy


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_P1-01F]

description: sci-RNA-seq3 library


title: SjS2_S4_tissue_biopsy

source_name: Lymph node groin

value: Lymph node groin

value: diagnostic


title: 21_13496PDMS206.CEL

value: multiple sclerosis

value: PBMC


title: WT, Input fraction, replicate 1

description:
WT, Input fraction, replicate 1
2935I

value: un-treated


title: WT, Light fraction, replicate 1

description:
WT, Light fraction, replicate 1
2935L


title: 21_8692PDMS206.CEL


title: 21_13382PDMS206.CEL


title: 21_13402PDMS206.CEL

value: female


title: 21_13497PDMS206.CEL


title: WT, Input fraction, replicate 2

description:
WT, Input fraction, replicate 2
2935I2


title: SjS2_S3_tissue_biopsy

source_name: Liver

value: Liver


title: SjS2_S2_tissue_biopsy

value: Pre eMZL diagnosis


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_P1-01D]

description: sci-RNA-seq3 library

value: lymphoblast

value: lentiviral transduction of a DDR-focused shRNA library


title: 21_13154PDMS206.CEL

value: male


title: WT, Heavy fraction, replicate 2

description:
WT, Heavy fraction, replicate 2
2935H2


title: WT, Heavy fraction, replicate 1

description:
WT, Heavy fraction, replicate 1
2935H


title: SjS2_S1_tissue_biopsy

source_name: Labial salivary gland

value: Labial salivary gland


title: SjS1_S2_tissue_biopsy

value: SjS eMZL


title: 21_13025TYMS206.CEL


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_P1-01B]

description: sci-RNA-seq3 library


description:
WT, Glucose starvation, Light fraction, replicate 1
2930L

value: kidney

value: Glucose starvation


title: SjS1_S1_tissue_biopsy


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-2_enrich_2_P1-12H]

description: sci-RNA-seq3 library


description:
WT, Glucose starvation, Input fraction, replicate 1
2930I

value: HEK293 TAF1 ts mutant cells


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_P1-01A]

description: sci-RNA-seq3 library


title: 21_12922PDMS206.CEL


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_P1-01C]

description: sci-RNA-seq3 library


title: RNA-Seq-siCMTR1-RKO

city: Xiamen

first: you

middle: a

last: bin

state: Fujian

address: 55 Zhenhai Road, Siming District, Xiamen

country: China

institute: the First Affiliated Hospital of Xiamen University

data_processing:
Basecalling was performed using CASAVA version 1.4
Reads were aligned to the hg19 genome assembly using Tophat version 2.1.1.
Assembly: hg19

source_name: RKO

growth_protocol: RKO cells were cultured in RPMI 1640 medium with 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin.

extract_protocol:
Total RNA was isolated using Ultrapure RNA Kit (CWBIO, Jiangsu, China) following the manufacturer’s protocol. DNase I in column digestion was included to ensure the RNA quality.
RNA library preparation was performed by using NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina (E7420L). Paired-end sequencing was performed with Illumina HiSeq platform.

treatment_protocol: RKO cells were transfected with the siRNA specifically targeting CMTR1 and maintained in 1640 medium supplemented with 10%FBS and 1% penicillin and streptomycin.

value: RKO

value: Human colorectal cancer cell line


title: RNA-Seq-siNC-RKO

value: siNC-RKO


description:
WT, Glucose starvation, Heavy fraction, replicate 1
2930H

value: WT


title: 21_12920PDMS206.CEL

value: multiple sclerosis

value: PBMC


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-2_enrich_2_P1-12F]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-2_enrich_2_P1-12E]

description: sci-RNA-seq3 library


description:
WT, Glucose starvation, Input fraction, replicate 2
2930I2


description:
WT, Glucose starvation, Heavy fraction, replicate 2
2930H2


title: 21_11078PDMS206.CEL


title: 21_11088PDMS206.CEL

value: female


description:
WT, Glucose starvation, Light fraction, replicate 2
2930L2


title: Female scramble control rep3 [scrF3]

city: Bronx

first: Blair

middle: Kathleen

last: Schneider

state: New York

address: 1300 Morris Park Ave., 809 Ullmann

department: Genetics

institute: Albert Einstein College of Medicine

data_processing:
HISAT2
DESeq2
Assembly: dm6
Supplementary files format and content: tab delimited text files contain all RPKM values for each sample and all genes

organism: Drosophila melanogaster

source_name: thoraces

extract_protocol: Trizol; Illumina; sequencing by synthesis (SBS) method

value: thoraces

value: adult day 20

tag: genetic background

tag: expression

value: scramble control


title: 21_11809TYMS206.CEL


title: Female scramble control rep2 [scrF2]


title: 20_7667PDMS204.CEL


title: 20_9874PDMS204.CEL


title: 20_9345PDMS204.CEL


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-2_enrich_2_P1-12B]

description: sci-RNA-seq3 library

value: lymphoblast

value: lentiviral transduction of a DDR-focused shRNA library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-2_enrich_2_P1-12A]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-2_enrich_2_P1-12G]

description: sci-RNA-seq3 library


title: 20_13431TYMS204.CEL

value: male


title: Female RooKD rep3 [rooF3]

value: RooKD


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-2_enrich_2_P1-11H]

description: sci-RNA-seq3 library


title: Female RooKD rep1 [rooF1]


title: Female RooKD rep2 [rooF2]


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-2_enrich_2_P1-11F]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-2_enrich_2_P1-11G]

description: sci-RNA-seq3 library


title: Female 412KD rep3 [F3_4121]


title: 20_12832PDMS204.CEL


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_P1-01E]

description: sci-RNA-seq3 library


title: 20_13234PDMS204.CEL


title: Female 412KD rep2 [F2_4121]


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-2_enrich_2_P1-12C]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-2_enrich_2_P1-11E]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-2_enrich_2_P1-11C]

description: sci-RNA-seq3 library


title: Female scramble control rep1 [scrF1]


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-2_enrich_2_P1-11D]

description: sci-RNA-seq3 library


title: 19_7949NMS202.CEL


title: RNAseq - WT34 - EpiLC

city: Leuven

first: Bernard

middle: Klaas

last: van der Veer

state: Vlaams-Brabant

address: Herestraat 49 - box 804

department: Department of Development and Regeneration

country: Belgium

institute: KU Leuven

data_processing:
Adapters, polyA/T tails, and bad quality reads (Phred score > 20) were trimmed using Trim Galore! (v0.6.4_dev) with default parameters. Reads were aligned to the transcriptome and quantified using Salmon (v0.14.1) (Patro et al., 2017) with default parameters using GENCODE release 23 of the mouse reference transcriptome sequences and the comprehensive gene annotation. Subsequently, the counts were imported into R (v4.0.2) using tximport (v1.18.0). Differentially expressed genes were defined using DEseq2 (v1.30.0) (Love et al., 2014) and log fold changes corrected using “ashr” method (FDR adjusted p.val < 0.05 & |log2(fold change)| > 1.5) (Stephens, 2017). Temporal differentially expressed genes were determined using xxx with default settings. GO term enrichment was performed using Cluster Profiler (3.18.1), while transcription factor enrichment was calculated using TRRUST website (https://www.grnpedia.org/trrust/) (Han et al., 2018). Lineage marker gene sets were determined by stratifying cell types of the mouse gastrulation reference dataset (Pijuan-Sala et al., 2019) per lineage and calculate markers function of Seurat (Hao et al., 2021). Single cell deconvolution was done with R package SCDC (v1.1.3) (Dong et al., 2021), using the two mixed gastrulation samples from the mouse gastrulation reference dataset as reference. TPM values were calculated using tximport.
Assembly: mm10
Supplementary files format and content: tab separated file with raw counts per sample
Supplementary files format and content: tab separated file with TPM normalized counts per sample

source_name: mESC B6129S6F1 Tet1<tm1Koh>

growth_protocol: Conversion of ESCs toward EpiLCs was performed as described previously by Hayashi et al. (2011), but without Activin A (Luo et al, 2020). EpiLCs are more recently characterized as a formative pluripotent state that models the E5-E6 pre-streak epiblast (7). Briefly, ESCs were first adapted for minimally five passages on to 2iL medium conditions on gelatin. 2iL media consists of basal N2B27 medium that consists of a 1:1 mixture of DMEM- F12 (Invitrogen, 11320-074) and Neurobasal medium (Invitrogen, 21103-049), 0.5 × N2 (Invitrogen, 17502-048), 0.5 × B27 (Invitrogen 17504-044), 2 mM L-glutamine, 0.1 mM 2-mercaptoethanol and 50:50 U:μg/mL penicillin/streptomycin), that is supplemented with 1 μM MEK inhibitor PD0325901, 3 μM GSK3 β inhibitor CHIR99021 and 0.01 ng/ml LIF (Peprotech, 250-02). Standard tissue culture plates were coated with 0.5 μg/cm2 fibronectin for 30 minutes and 3.0 × 104 cells/cm2 were seeded in EpiLC conversion medium, which consists of basal N2B27 medium supplemented with 1% knockout serum replacement (KOSR) and 12 ng/mL bFGF. At day 1, medium was changed and cells were considered to be EpiLCs after 2 days of culturing.

extract_protocol:
Total RNA was extracted from cells using Trizol using the manufacturer’s instructions.
RNA sequencing (RNAseq) libraries were prepared from 4 µg of total RNA using the KAPA stranded mRNA-seq kit (Roche, KK8421) according to manufacturer’s specifications. 100 nM KAPA-single index adapters (Roche, KK8700) were added to A-tailed cDNA, and libraries were amplified for 10 cycles. Finally, 1x library cleanup was performed using Agencourt AMPure XP beads (Beckman Coulter, A63881). Library fragment size was assessed using Agilent Bioanalyzer 2100 with the High Sensitivity DNA analysis kit (Agilent, 5067-4626) and concentration was determined using Qubit™ dsDNA HS Assay kit (Invitrogen, Q32854). Each library was diluted to 4 nM and pooled for sequencing on an Illumina Hiseq4000, aiming at 15-20 million SE50 reads per sample (19 million reads on average).

value: mESC B6129S6F1 Tet1<tm1Koh>

value: mESC

value: EpiLC


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-2_enrich_2_P1-11B]

description: sci-RNA-seq3 library


title: 20_10667TYMS204.CEL


title: 20_10461PDMS204.CEL


title: 19_7169RRMS202.CEL


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-2_enrich_2_P1-11A]

description: sci-RNA-seq3 library


title: RNAseq - WT17 - EpiLC


title: 19_6712RRMS202.CEL


title: 19_620NMS202.CEL


title: RNAseq - WT12 - EpiLC


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-2_enrich_2_P1-10H]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-2_enrich_2_P1-10G]

description: sci-RNA-seq3 library


title: Female 412KD rep1 [F1_4121]


title: 19_13416NMS202.CEL


title: RNAseq - KO9 - EpiLC

value: Tet1 KO


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-2_enrich_2_P1-10F]

description: sci-RNA-seq3 library


title: RNAseq - KO25 - EpiLC


title: RNAseq - KO15 - EpiLC


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-2_enrich_2_P1-10D]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-2_enrich_2_P1-10E]

description: sci-RNA-seq3 library


title: 19_12930NMS202.CEL


title: 19_13009NMS202.CEL


title: 19_12903NMS202.CEL


title: ATACseq -KO15 - day 3 + XAV

description:
KO_X_d3.pooled.fc.signal.bigwig
KO_X_d3vsWT_d3.log2.signal.bigwig
antNPC.d3.DOR.KOvsWT.open.order.asc.bed
antNPC.d3.DOR.KOvsWT.close.order.asc.bed

data_processing:
ATACseq reads were analyzed using ENCODE ATACseq pipeline (http://doi.org/10.5281/zenodo.156534) developed by Anshul Kundaje’s laboratory, which performs quality and adapter trimming, alignment, deduplication, peak calling and quality control in a fully automated manner. The resulting peak files and alignment files were used by DiffBind (v3.0.15) to find differentially accessible regions (DARs) using settings specific for ATACseq; “summits = 150” in peak defining and “background = true” in read normalization. Transcription factor motif enrichment in DARs was determined using HOMER (v2) package with default settings. Profile plots and heatmaps were made using DeepTools (v3.4.3) from bigwig files generated from merged replicate reads with the ATACseq pipeline.
Assembly: mm10
Supplementary files format and content: bam file with aligned and deduplicated reads
Supplementary files format and content: narrowPeak file with peaks per sample
Supplementary files format and content: bigwig file per sample
Supplementary files format and content: bigwig file for pooled reads
Supplementary files format and content: bigwig file for log2 fold change for pooled reads (only some files)
Supplementary files format and content: bed file with differentially accesbile reagions (open)
Supplementary files format and content: bed file with differentially accesbile reagions (close)

growth_protocol: Anterior neural progenitor differentiation was performed as previously described (Cruz-Molina et al., 2017; Gouti et al., 2014; Luo et al., 2020). Feeder depleted serum cultured ESCs were plated on gelatin coated plates at 10,000 cells/cm2 (90,000 cells per well in a 6-well plate) in advanced N2B27 defined media compromising a 1:1 mix of 1:1 DMEM/F12 (Invitrogen, 12634-010, Invitrogen) and Neurobasal medium (Invitrogen, 10888-022) supplemented with 0.5x B27 without vitamin A (Invitrogen, 12587-010), 0.5x N2 (Invitrogen, 17502-048), 2 mM L-glutamine (Invitrogen, 25030-024), 40 mg/ml BSA fraction V (Invitrogen, 15260037), 0.1 mM 2-mercaptoethanol (Invitrogen, 31350-010) and 100:100 units:µg/ml penicillin:streptomycin (Sigma-Aldrich, P4333; or Gibco, 15140122). Cells were supplemented with 10 ng/ml bFGF (Cat#100-18C, Peprotech) from day 0 until day 3. At day 3 the medium was changed to medium without bFGF. In some experiments, 5 µM of the Wnt inhibitor XAV939 (Sigma-Aldrich, X3004), 2.5 µM of the TGF-β receptor inhibitor SB431542 (Sigma-Aldrich, S4317), 0.1 µM BMP inhibitor LDN193189 (Sigma-Aldrich, SML0559), 0.3 µM or 3 μM GSK3 β inhibitor CHIR99021 (Axon Medchem BV,1408), 5 or 25 ng/ml of Activin A (Peprotechm 120-14E), or equivalent amount of DMSO (vehicle control) were added from day 2 until day 5. The medium was changed every day during differentiation, except on day 1. At day 4, 1.5x amount of medium was added. The experimental end-point is day 5.

extract_protocol:
Although viability of the cells is high (> 90-95%), we treated them in suspension nonetheless with 0.1 mg/ml DNase I (Worthington, LS002005) for 30 minutes at 37 ºC directly in the medium and added digestion buffer (0.5 mM CaCl2, 2.5 mM MgCl2). Cells were washed with PBS and incubated for 5 minutes with TrypLE which was subsequently quenched with medium.
We used the omni-ATAC protocol to prepare ATACseq libraries as described before (Buenrostro et al., 2013; Corces et al., 2017).  After two washes with ice-cold PBS, 50,000 cells were pelleted in a pre-cooled 1.5 mltube by centrifugation for 5 minutes at 600 rcf at 4 ºC in a microcentrifuge. Supernatant was removed and 50 µl of ATAC-lysis buffer was added (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% NP40, 0.1% Tween-20, 0.01% digitonin) and incubated on ice for 3 minutes, followed by addition of 1 ml of ATAC-wash buffer (ATAC-lysis buffer, without NP40 and digitonin) and the tube was inverted 3 times. The nuclei were pelleted for 10 minutes at 600 rcf at 4 ºC in a microcentrifuge. Supernatant was removed, and nuclei were resuspended in 50 µl transposition reaction buffer (5 µl H2O, 15 µl PBS, 0.5 µl 10% Tween-20, 0.5 µl 1% digitonin, 25 µl TD buffer, 2.5 µl Tagment DNA Enzyme 1) (Tagment DNA Enzyme and Buffer Small Kit, Illumina, 20034197) and incubated for 30 minutes at 37 ºC at 1000 rpm in a thermomixer. The transposed DNA was cleaned up immediately following transposition ZymoDNAClean& Concentrator-5 kit (Zymo D4014) following manufacturer instructions. The transposed DNA was amplified in one pre-amplification step (5 cyles) and a final amplification (5 to 7 cycles) in the same 50 µl reaction using 1x NEBNext High-fidelty PCR master mix (Bioké/NEB, M0541S) and 125 nM of Ad1_noMX primer and Ad2.index primer with the following program: 72 ºC 5 min, 98 ºC 30 sec; 5 to 7x 98 ºC 10 sec, 63 ºC 30 sec, 72 ºC 1 min; 72 ºC 1 min. For each library, the optimal amplification was optimized using qPCR; 1/5th of the pre-amplified libraries was added to amplification master mix containing SYBR Green and run on a Applied Biosystems StepOnePlus Real-Time PCR system  for an additional 20 cycles. Relative log-fluorescence vs amplification cycle was plotted out to manually determine the appropriate amplification cycles. The number of cycles was selected to reach 1/3 of the final relative fluorescence unit value. Following amplification, the libraries were purified using ZymoDNAClean& Concentrator-5 kit (Zymo D4014) followed by 0.55x-1.75x dual size slection using AMPure XP beads. The quality of the libraries was assessed using an Agilent Bioanalyzer 2100 with the High Sensitivity DNA analysis kit (Agilent, 5067-4626) and concentration was determined using Qubit™ dsDNA HS Assay kit (Invitrogen, Q32854). The libraries were diluted to 4 nM and pooled together for SE50 sequencing on an Illumina Hiseq4000, aiming for minimally 25 million mapped and unique reads.
SE50

value: day 3 + XAV

tag: molecule type

value: transposed DNA


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-2_enrich_2_P1-10B]

description: sci-RNA-seq3 library


title: RNAseq - WT7 - EpiLC

value: WT


title: 19_11206NMS202.CEL

value: multiple sclerosis


title: ATACseq -KO1 - day 3 + XAV


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-2_enrich_2_P1-10A]

description: sci-RNA-seq3 library


title: 18_8265NMS201.CEL

value: PBMC


title: 18_4397NMS201.CEL

value: female


title: 19_12905NMS202.CEL


title: 18_13054NMS201.CEL


title: Spleen, OFF, rep1

city: Toronto

first: Juan Carlos

last: Zuniga-Pflucker

state: Ontario

address: 2075 Bayview Avenue

country: Canada

institute: University of Toronto

data_processing:
Samples were aligned to GRCm38 mouse genome. Next, the samples were filtered of genes that were not expressed across all samples and gene expression was normalized.
Unsupervised clustering of the samples and statistically significant differential expression analysis was performed on the samples where p<0.05 was considered significant. All data analysis was performed using R software version 3.6.2 using edgeR and enrichGO.

source_name: Spleen

extract_protocol:
Spleens were harvested from WT and RBPJind mice and RBPJind INT cells (CD3+CD4+CD25-CD45RBint) and WT MNT cells (CD3+CD4+CD25-CD45RBhi) were FACS sorted.
The cells were placed in Trizol and their RNA extracted. The RNA samples were delivered to the University Health Network Bioinformatics and HPC Core where the samples were sequenced using Illumina Novaseq 6000 and the samples aligned to GRCm38 mouse genome.

treatment_protocol: RBPJind mice were induced with Dox water for a period of 4 weeks to generate RBPJind immature naïve T (INT) cells that had matured in the presence of Notch signals or induced with Dox for 3 weeks followed by 1 week without Dox during their peripheral maturation.

value: Spleen

value: Spleen CD3+CD4+CD25-CD45RBint

value: RBPJind

value: Dox


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-2_enrich_2_P1-09H]

description: sci-RNA-seq3 library

value: lymphoblast

value: lentiviral transduction of a DDR-focused shRNA library


title: ATACseq -WT34 - day 3 + CHIR

description:
WT_C_d3.pooled.fc.signal.bigwig
WT_C_d3vsWT_d3.log2.signal.bigwig
antNPC.d3.DOR.KOvsWT.open.order.asc.bed
antNPC.d3.DOR.KOvsWT.close.order.asc.bed

value: day 3 + CHIR


title: RNAseq - KO1 - EpiLC

value: mESC B6129S6F1 Tet1<tm1Koh>


title: ATACseq -WT7 - day 3 + CHIR

value: mESC


title: Spleen, ON, rep2


title: Spleen, ON, rep1


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-2_enrich_2_P1-10C]

description: sci-RNA-seq3 library


title: ATACseq - WT72 - day 3

description:
WT_d3.pooled.fc.signal.bigwig
antNPC.d3.DOR.KOvsWT.open.order.asc.bed
antNPC.d3.DOR.KOvsWT.close.order.asc.bed

value: day 3


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-2_enrich_2_P1-09F]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-2_enrich_2_P1-09G]

description: sci-RNA-seq3 library


title: Spleen, WT, rep2

value: Spleen CD3+CD4+CD25-CD45RBhi


title: 18_13044NMS201.CEL

value: male


title: Spleen, OFF, rep2


title: ATACseq -WT72 - day 2

description:
WT_d2.pooled.fc.signal.bigwig
antNPC.d3.DOR.KOvsWT.open.order.asc.bed
antNPC.d3.DOR.KOvsWT.close.order.asc.bed

value: day 2

value: transposed DNA


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-2_enrich_2_P1-09E]

description: sci-RNA-seq3 library


title: Spleen, WT, rep1

value: WT


title: 18_13042NMS201.CEL

value: multiple sclerosis

value: PBMC


title: 18_12880NMS201.CEL

value: female


title: 18_11912NMS201.CEL


title: ATACseq - WT70 - day 3


title: ATACseq -WT70 - day 2


title: Naïve, OFF, rep2

data_processing:
Raw fastq files were analyzed using Galaxy ATAC-Seq data analysis protocol.
Trimmed sequences were mapped to mm10 genome using Bowtie2 and MACS2 was used for peak calling with nomodel, 200-bp extension size, and 100-bp shift size.
Only peaks called with a minimum q-value of 0.05 were selected and peaks were compared and visualized using Integrated Genome Browser.
Supplementary files format and content: bigWig files for visualizing the peaks.

extract_protocol:
Spleens were harvested from WT and RBPJind mice and RBPJind INT cells (CD3+CD4+CD25-CD45RBint) and WT MNT cells (CD3+CD4+CD25-CD45RBhi) were FACS sorted.
Cells were resuspended in transposition reaction mix (25 μl TD, 2.5 μl TDE1, and 22.5 μl Nuclease Free Water) followed by 20 min incubation at 37˚C degrees. Qiagen MinElute PCR Purification Kit was used to purify transposed DNA and DNA samples were sent to Princess Margaret Genomics Centre for downstream PCR amplification and sequencing.

value: Spleen

value: Spleen CD3+CD4+CD25-CD45RBint


title: 17_8429RR162.CEL


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-2_enrich_2_P1-09B]

description: sci-RNA-seq3 library


title: ATACseq - KO15 - day 3

description:
KO_d3.pooled.fc.signal.bigwig
KO_d3vsWT_d3.log2.signal.bigwig
antNPC.d3.DOR.KOvsWT.open.order.asc.bed
antNPC.d3.DOR.KOvsWT.close.order.asc.bed

value: Tet1 KO


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-2_enrich_2_P1-09C]

description: sci-RNA-seq3 library

value: lymphoblast


title: Naïve, OFF, rep1

value: RBPJind

value: Dox


title: Naïve, ON, rep2


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-2_enrich_2_P1-09A]

description: sci-RNA-seq3 library

value: lentiviral transduction of a DDR-focused shRNA library


title: ATACseq - KO15 - day 2

description:
KO_d2.pooled.fc.signal.bigwig
antNPC.d3.DOR.KOvsWT.open.order.asc.bed
antNPC.d3.DOR.KOvsWT.close.order.asc.bed

value: mESC B6129S6F1 Tet1<tm1Koh>

value: mESC


title: 17_7708RRMS200.CEL


title: ATACseq - KO1 - day 3

value: day 3


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-2_enrich_2_P1-08H]

description: sci-RNA-seq3 library


title: Naïve, ON, rep1


title: 17_13235NMS200.CEL


title: Naïve, WT, rep2

value: Spleen CD3+CD4+CD25-CD45RBhi


title: ATACseq - KO1 - day 2

value: day 2


title: ATACseq - CM69 - day 3

description:
CM_d3.pooled.fc.signal.bigwig
antNPC.d3.DOR.KOvsWT.open.order.asc.bed
antNPC.d3.DOR.KOvsWT.close.order.asc.bed

source_name: mESC B6129S6F1 Tet1<tm1Koh>; CRISPR HDR

value: mESC B6129S6F1 Tet1<tm1Koh>; CRISPR HDR

value: Tet1 MUT

value: transposed DNA


title: Naïve, WT, rep1

value: WT


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-2_enrich_2_P1-08F]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-2_enrich_2_P1-09D]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-2_enrich_2_P1-08G]

description: sci-RNA-seq3 library


title: ATACseq - CM69 - day 2

description:
CM_d2.pooled.fc.signal.bigwig
antNPC.d3.DOR.KOvsWT.open.order.asc.bed
antNPC.d3.DOR.KOvsWT.close.order.asc.bed


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-2_enrich_2_P1-08E]

description: sci-RNA-seq3 library


title: Bone marrow, fracture and TBI, rep3 [WT_3]

city: Wuhan

first: Liu

last: Weijian

address: Jiefang Avenue

institute: Union hospital

data_processing:
The quality control of RNASeq rawdata was performed by FastQC (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/) software.
Then sequence quality was also verified using fastp
Hisat2 (http://daehwankimlab.github.io/hisat2/) was employed to map the cleaned reads to reference genome mm10.
Transcriptomes from all samples were merged to reconstruct a comprehensive transcriptome using gffcompare (https://github.com/gpertea/gffcompare/)
StringTie and was used to estimate the expression levels of all transcripts. StringTie was used to perform expression level for mRNAs by calculating FPKM
The differentially expressed mRNAs were selected by R package edgeR (https://bioconductor.org/packages/release/bioc/html/edgeR.html)
Assembly: mm10
Supplementary files format and content: tab-delimited text files include FPKM values for each Sample

source_name: Bone marrow

extract_protocol:
RNA was harvested using Trizol (Invitrogen, Carlsbad, CA, USA).
RNA libraries for RNA-seq were prepared using SMARTER mRNA-Seq Library Prep Kit following manufacturer's protocols.

value: Bone marrow

value: Wild type

value: Fracture and TBI


title: 17_13143NMS200.CEL

source_name: PBMC

value: multiple sclerosis

value: PBMC

value: female


title: Bone marrow, fracture and TBI, rep2 [WT_2]


title: Bone marrow, fracture and TBI, rep1 [WT_1]


title: 17_13059NMS200.CEL

value: male


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-2_enrich_2_P1-08D]

description: sci-RNA-seq3 library

data_processing:
For the sci-RNA-seq3 transcriptome libraries, sequencing reads from the Illumina NextSeq platforms were first demultiplexed based on PCR index using the bcl2fastq software. Reads were filtered based on RT and ligation index, by allowing <2 hamming distance from reference. Filtered reads were trimmed with TrimGalore (v0.6.6) with parameters: -a AAAAAAAA --three_prime_clip_R1 1. Reads were then aligned to the GRCh38 reference genome using STAR (v2.7.6a). PCR duplicates were collapsed using the RT index, ligation index, UMI sequence and end coordinate of reads. Reads were further demultiplexed based on the combination of the RT, ligation and PCR index and split into files for individual cells. To generate gene expression count matrices, reads were assigned to the exonic and intronic region of closest genes with HTseq (v.2.0.2). Reads with ambiguous assignments were discarded. Cells were further filtered based on total UMI (> 100) and mitochondrial reads percentage (<10%). Cells with the number of features detected between 10% and 90% percentile of all cells were kept and considered high-quality cells. The single cell analysis was performed using the Seurat (v4.0.0) package in R.
For capture libraries of the synHEK3 and shRNA constructs, sequencing reads from the Illumina NextSeq platforms were first demultiplexed based on PCR index using the bcl2fastq software. A custom script was used to further demultiplex and filter the synHEK3 and shRNA libraries based on the combination of RT, ligation and PCR index (cell ID), by allowing <2 hamming distance from reference. For the synHEK3 library, reporter barcodes (<2 hamming distance to the reference barcode set), prime editing outcomes and read UMIs were extracted. For the shRNA library, shRNA sequences (<2 hamming distance to the reference barcode set) and read UMIs were extracted. UMIs <3 hamming distance were collapsed.   A series of pre-processing steps were applied to the data. 1) We matched cell IDs between the sci-RNA-seq3 transcriptome libraries and the capture libraries, and only kept high-quality cells (number of features between 10% and 90% percentile). 2) For the shRNA library, cells with <3 or >200 UMIs or >20 shRNA were removed. 3) For the synHEK3 library, cells with <4 UMIs were first removed. We counted UMIs for synHEK3 reporters belonging to the two clones and calculated a clone UMI/total UMI ratio. If the ratio was >80% for a specific clone, the cell is assigned to the corresponding clone. Cells with extremely high UMIs (cutoff between 100 and 250 UMIs, depending on library complexity and sequencing depth of the plate) were also removed for downstream analysis. 4) For a cell-synHEK3 barcode combination, if multiple insertion sequences were observed, sequences <3 hamming distance were collapsed. Cell-synHEK3 barcode pairs having conflicting editing outcomes were discarded. 5) A cell-synHEK3 barcode-editing outcome-shRNA matrix was generated for a common set of cells identified by the shRNA and synHEK3 libraries.
Assembly: GRCh38
Supplementary files format and content: cell_synHEK3_shRNA.csv.gz contains a cell-synHEK3-shRNA capture matrix

extract_protocol: We performed a modified version of sci-RNA-seq3 (Martin et al., 2022). In brief, K562 cells were lysed with 5 mL of a hypotonic, PBS-based lysis buffer containing DEPC. The resulting nuclei were fixed with 4 volume of ice-cold methanol. After rehydrating and washing the nuclei carefully in a sucrose/PBS/triton buffer (SPBST), the nuclei were treated with T7 polymerase in an NTP mix buffer at 37oC for 30 min. Then nuclei were washed, sonicated briefly and counted. The nuclei were distributed to a 96-well plate for reverse transcription, with a cocktail of three indexed primers: oligo-dT, HEK3 and CS1 -- specific for the cellular transcriptome, and RNAs produced from the synHEK3 reporter and the shRNA constructs, respectively. After reverse transcription, nuclei were pooled, washed in SPBST and redistributed to 4 fresh plates for ligation of the second index primer with T4 DNA ligase. Nuclei were then again pooled, washed, and redistributed to 4 final plates at two different concentrations (1300 cell per well for 2 plates and 2500 cell per well for 2 plates) for second strand synthesis and extraction. Nuclear lysates were split in half, with one half undergoing normal sci-RNA-seq3 transcriptome library preparation (tagmentation and PCR to add the third index) and the other half subjected to indexed enrichment PCR for both the synHEK3 and shRNA constructs. Products were pooled by PCR plate, size-selected and sequenced on an Illumina Nextseq 2000 or Nextseq 550.

value: lymphoblast

value: lentiviral transduction of a DDR-focused shRNA library


title: 17_12907NMS200.CEL


title: ATACseq - CM59 - day 2

value: mESC


title: WT70 - day5

description:
antNPCs_F1Tm1_3_counts.csv
antNPCs_F1Tm1_3_TPM.csv

extract_protocol:
Total RNA was extracted from cells using Trizol using the manufacturer’s instructions.
RNA sequencing (RNAseq) libraries were prepared from 4 µg of total RNA using the KAPA stranded mRNA-seq kit (Roche, KK8421) according to manufacturer’s specifications. 100 nM KAPA-single index adapters (Roche, KK8700) were added to A-tailed cDNA, and libraries were amplified for 10 cycles. Finally, 1x library cleanup was performed using Agencourt AMPure XP beads (Beckman Coulter, A63881). Library fragment size was assessed using Agilent Bioanalyzer 2100 with the High Sensitivity DNA analysis kit (Agilent, 5067-4626) and concentration was determined using Qubit™ dsDNA HS Assay kit (Invitrogen, Q32854). Each library was diluted to 4 nM and pooled for sequencing on an Illumina Hiseq4000, aiming at 15-20 million SE50 reads per sample (19 million reads on average).
SE50

value: mESC B6129S6F1 Tet1<tm1Koh>


title: 17_13159NMS200.CEL


title: Bone marrow, fracture and TBI, rep3 [Adrb3_KO_3]

value: Adrb3 knockout


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-2_enrich_2_P1-08B]

description: sci-RNA-seq3 library


title: Bone marrow, fracture and TBI, rep2 [Adrb3_KO_2]


title: 17_13165NMS200.CEL


title: WT70 - day3


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-2_enrich_2_P1-08A]

description: sci-RNA-seq3 library


title: 16_9959RRMS199.CEL


title: 16_8551NMS161.CEL


title: CM69 - day5


title: Bone marrow, fracture and TBI, rep1 [Adrb3_KO_1]


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-2_enrich_2_P1-07H]

description: sci-RNA-seq3 library


title: 16_7926NMS199.CEL


title: Bone marrow, fracture and TBI, rep3 [Adrb2_KO_3]

value: Adrb2 knockout


title: CM69 - day3

value: mESC B6129S6F1 Tet1<tm1Koh>; CRISPR HDR


title: ATACseq - CM59 - day 3

value: Tet1 MUT

value: day 3


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-2_enrich_2_P1-07G]

description: sci-RNA-seq3 library


title: 17_12859NMS200.CEL


title: Bone marrow, fracture and TBI, rep2 [Adrb2_KO_2]


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-2_enrich_2_P1-08C]

description: sci-RNA-seq3 library


title: CM59 - day5


title: 16_7274RRMS199.CEL


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-2_enrich_2_P1-07F]

description: sci-RNA-seq3 library


title: 16_602NMS199.CEL


title: Bone marrow, fracture and TBI, rep1 [Adrb2_KO_1]


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-2_enrich_2_P1-07E]

description: sci-RNA-seq3 library


title: NWD1_B

city: BRONX

first: Leonard

last: Augenlicht

address: 1300 Morris Park Avenue

department: Cell Biology

institute: ALBERT EINSTEIN COLLEGE OF MEDICINE

description: chromatin accessibility

data_processing:
cellranger-atac pipeline 2.1.0
Assembly: mm10
Supplementary files format and content: HDF5 Feature Barcode Matrix Format

source_name: intestinal epithelial cells

growth_protocol: Bl6 mice were raised up under normal condition

extract_protocol:
single nuclei extration
Our single cell ACTAseq (scATACseq) libraries were made according to the manufacturer’s protocol for Chromium Next scATAC kit.2

treatment_protocol: After weaning, mice were fed with AIN or NWD1 for 3 months.

value: intestinal epithelial cells

tag: cell marker

value: Epcam +, Cd45 -

value: NWD1 feeding


title: CM59 - day3

tag: genotype


title: WT34 (timecourse) - day4

description:
antNPCs_F1Tm1_2_counts.csv
antNPCs_F1Tm1_2_TPM.csv

value: WT


title: 16_5779NMS199.CEL

value: multiple sclerosis


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-2_enrich_2_P1-07D]

description: sci-RNA-seq3 library


title: 16_13008NMS199.CEL

value: PBMC


title: NWD1_A

description: chromatin accessibility


title: 16_12866NMS199.CEL

value: female


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-2_enrich_2_P1-07C]

description: sci-RNA-seq3 library


title: AIN_A

description: chromatin accessibility

value: AIN feeding


title: WT34 (timecourse) - day2


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-2_enrich_2_P1-07B]

description: sci-RNA-seq3 library

value: lymphoblast


title: WT34 (timecourse) - day1


title: 16_11114NMS199.CEL


title: WT7 (timecourse) - day5


title: AIN_B

description: chromatin accessibility


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-2_enrich_2_P1-07A]

description: sci-RNA-seq3 library

value: lentiviral transduction of a DDR-focused shRNA library


title: 16_11104NMS199.CEL

value: male


title: Sca1_AAV_Malat1

city: Hangzhou

first: Lingxia

last: Lyu

address: No.79 Qingchun Road

institute: Zhejiang university

description: 10x Genomics

library_source: transcriptomic single cell

data_processing:
Data were processed following standard protocols using Cell Ranger version 3.1.0.
Cellranger mkfastq was used to demultiplex raw base call files into fastq files.
Cellranger count was used to perform alignment, filtering, barcode counting, UMI counting, and to generate feature-barcode matices.
Assembly: mm10
Supplementary files format and content: Tab-separated values files and matrix files

source_name: veingraft

extract_protocol:
Veingrafts were harvested and digested. After full digestion, cell pellets were suspended in PBS and stained with a LIVE/DEAD ™ Fixable Near-IR Dead Cell Stain Kit (1:1000) and Hoechst 33342 (Invitrogen, H3570, 1:1000). After washing using PBS, cells were resuspended in PBS, after which single nucleated live cells (Hoechst+ & Dead Cell Stain-) were sorted into PBS with 0.04% BSA using a BD FACS ARIA II Flow Cytometer (BD Biosciences). For CD45- cells, a CD45 antibody (Invitrogen, 14-0452-82, 1:2000) was additionally used. For RFP+ cells, FACS sorting was based on their autofluorescence. Samples were subjected to scRNA-seq.
A ChromiumTM Single Cell 3' Reagent Kit v2 or v3 chemistry (10x Genomics) was used and a standard protocol followed. The library was generated and sequenced on a Novaseq6000 PE150 platform (Illumina) with paired-end 150 bp sequencing strategy.

value: veingraft

value: Goblet cells

value: all cells

value: Sca1 creER;R26-tdtomato


title: WT7 (timecourse) - day4

value: mESC B6129S6F1 Tet1<tm1Koh>

value: mESC


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-2_enrich_2_P1-12D]

description: sci-RNA-seq3 library


title: 16_11081NMS199.CEL


title: Sca1_RFP_CD45

description: 10x Genomics

value: No


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-2_enrich_2_P1-06F]

description: sci-RNA-seq3 library


title: WT34 (timecourse) - day3


title: WT7 (timecourse) - day2


title: 16_10956NMS199.CEL


title: 16_10924NMS199.CEL


title: 15_13163NMS198.CEL


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-2_enrich_2_P1-06E]

description: sci-RNA-seq3 library