title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_TX_P1-06E]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_TX_P1-06B]

status: Public on Apr 12 2023

type: SRA

city: Seattle

first: Xiaoyi

last: Li

state: WA

address: 3720 15th Ave NE

department: Department of Genome Sciences

country: USA

institute: University of Washington

description: sci-RNA-seq3 library

library_source: transcriptomic

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

organism: Homo sapiens

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_Plate2500-2_TX_P1-06A]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_TX_P1-05A]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_TX_P1-05B]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_TX_P1-05F]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_TX_P1-05E]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_TX_P1-04H]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_TX_P1-05C]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_TX_P1-07B]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_TX_P1-05D]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_TX_P1-06G]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_TX_P1-04D]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_TX_P1-04G]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_TX_P1-03H]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_TX_P1-04E]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_TX_P1-03D]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_TX_P1-04A]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_TX_P1-04B]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_TX_P1-05G]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_TX_P1-04C]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_TX_P1-02F]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_TX_P1-03F]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_TX_P1-03A]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_TX_P1-02E]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_TX_P1-02C]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_TX_P1-02G]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_TX_P1-02D]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_TX_P1-02B]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_TX_P1-02A]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_TX_P1-01E]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_TX_P1-01G]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_TX_P1-02H]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_TX_P1-04F]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_TX_P1-01F]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_TX_P1-03G]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_TX_P1-03E]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_TX_P1-01B]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_TX_P1-03C]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_TX_P1-03B]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_TX_P1-01C]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_TX_P1-01H]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_2_P1-12F]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_2_P1-12E]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_TX_P1-01A]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_2_P1-12C]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_2_P1-12B]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_2_P1-12G]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_2_P1-12D]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_2_P1-12A]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_2_P1-11H]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_2_P1-11D]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_2_P1-11G]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_2_P1-11E]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_2_P1-11A]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_2_P1-10H]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_2_P1-11C]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_2_P1-11F]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_2_P1-10G]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_TX_P1-01D]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_2_P1-10E]

description: sci-RNA-seq3 library


title: Prime editing library of syHEK3 reporters in CRISPRoff treated cells [K562-SC1-5-OFF-USP7-Rep2]

description:
synHEK3 amplicon sequencing library
CRISPRoff_USP7.csv

library_source: genomic

data_processing:
T7-assisted reporter mapping: Sequencing reads were first demultiplexed using the bcl2fastq software. Under the current library design and sequencing scheme, Read1 started from genomic sequence and extended into the integrated synHEK3 construct, while Read2 contained reporter barcode information. Therefore, 1) for each sequencing read pair, the 16-bp reporter barcode was extracted from Read 2 and attached to the read name of Read 1. 2) Read1 was trimmed using cutadapt (v4.1) with following parameters: --cores=4 --discard-untrimmed -e 0.2 -m 10 -O 8 -a CCCTAGAAAGATAGTCTGCGTAAAATTGACGCATG. The adapter corresponded to the 3’ITR of piggyBac transposon. Since parameter “--discard-untrimmed” was used, only reads spanning insertion junctions were kept and trimmed. 3) Trimmed sequences were aligned to the GRCh38 reference genome using bwa mem (v0.7.17). The “-Y” option was used to enable soft clipping for supplementary alignments. 4) Reads uniquely (without XA:Z tag) and contiguously mapped near putative piggyBac landing pads (TTAA motifs) were kept using samtools (v1.9) and a custom script. 5) Aligned reads were converted to BED format using the sam2bed function in bedops (v2.4.35). Reads aligned to standard chromosomes were kept. 6) Insertion points were calculated for all reads based on strand of alignment. And reads were sorted by the insertion coordinates. 7) The first 8-bp of reads were used as UMIs. A custom script was used to collapse reads at a per-location, per-barcode, per UMI basis. A barcode-location-UMI count table was generated. 8) synHEK3 barcodes <3 Levenshtein Distance at each location were collapsed and barcodes with > 3 UMIs were kept. 9) The count table was converted to a GenomicRanges object in R. Coordinates of the last 4 base pairs of aligned reads were designated as genomic locations of the inserted synHEK3 reporters. 10) “Landing pads” of the mapped synHEK3 reporters were retrieved using the getSeq() function in the BSgenome package. Reporters that didn’t have a TTAA sequence (which could be due to PCR error, mapping error, or the use of non-canonical landing pads) were removed. 11) SynHEK3 barcodes mapped to more than one location were removed.
SynHEK3 amplicon sequencing library: Sequencing reads from the Illumina NextSeq platforms were first demultiplexed using the bcl2fastq software. The 16-bp reporter barcodes were extracted from read and attached to its read name. Sequences around the CRISPR cut site were extracted for all reads. A barcode-editing outcome table was generated. For prime editing experiments, a custom script was used to align sequences to a reference sequence and count mutation frequency for every barcode. For Cas9 mutagenesis analysis, all sequences were aggregated and editing outcomes (alleles) with the highest number of counts were selected. These most frequent alleles were then aligned to reference sequences using needleall with the following parameters: -gapopen 20 -gapextend 0.5 -endopen 20. A custom script was used to annotate the mutational events.
Amplicon sequencing of endogenous genes: Sequencing reads were demultiplexed using the bcl2fastq software (Illumina). A custom script was used to determine frequencies of the introduced substitutions.
Assembly: GRCh38
Supplementary files format and content: T7-500-1_S1_ITR_corrected.txt contains processed, barcode collapsed synHEK3 integration raw counts
Supplementary files format and content: T7-500-1_processed.csv contains fully processed, unique synHEK3 reporters and relevent information
Supplementary files format and content: K562-HEK3-insCTT-DMSO_count.txt contains processed prime editing outcomes of all synHEK3 barcodes
Supplementary files format and content: K562-HEK3-Cas9-Day1-XL281_S68_barcode_edit.txt contains processed Cas9 indel frequency of all synHEK3 barcodes on Day 1
Supplementary files format and content: K562-HEK3-Cas9-Day2-XL281_S70_barcode_edit.txt contains processed Cas9 indel frequency of all synHEK3 barcodes on Day 2
Supplementary files format and content: K562-HEK3-Cas9-Day4-XL281_S72_barcode_edit.txt contains processed Cas9 indel frequency of all synHEK3 barcodes on Day 4
Supplementary files format and content: sg1_top_alleles_count.txt contains Cas9 editing allele count for all synHEK3 barcodes on Day 4
Supplementary files format and content: sg1_top_alleles_info.txt contains annotations of the most frequent alleles for Cas9 editing on Day 4
Supplementary files format and content: CRISPRoff_LRRC8C.csv contains processed prime editing frequencies of synHEK3 reporters in the CRISPRoff experiment, LRRC8C vs. non-targeting control
Supplementary files format and content: CRISPRoff_METTL2A.csv contains processed prime editing frequencies of synHEK3 reporters in the CRISPRoff experiment, METTL2A vs. non-targeting control
Supplementary files format and content: CRISPRoff_USP7.csv contains processed prime editing frequencies of synHEK3 reporters in the CRISPRoff experiment, USP7 vs. non-targeting control
Supplementary files format and content: CRISPRa_CXCR4_IL2RB_EGFR_CDKL5.csv contains prime editing frequencies at endogenous loci before and after CRISPR activation
Supplementary files format and content: shrna_freq_table.csv contains prime editing frequencies of synHEK3 reporters measured in cells transduced with individual shRNAs (GFP positive and GFP negative)

molecule: genomic DNA

extract_protocol:
gDNA of K562 cells was purified using the DNeasy Blood & Tissue Kit. Alternatively, cells were lysed in a lysis buffer [100 mM Tris-HCl pH8.0, 0.05% SDS and 0.04 mg/mL proteinase K(Thermo Scientific)], and incubated at 50 oC 60 min and 80 oC 30 min.
T7-assisted reporter mapping: gDNA of K562 cells was purified using the DNeasy Blood & Tissue Kit (QIAGEN) and in vitro transcribed with the HiScribe T7 High Yield RNA Synthesis Kit (New England Biolabs). Briefly, each reaction (20 uL) contained 0.3~1 ug gDNA, NTPs (10 mM each) and 2 ul T7 RNA Polymerase Mix. The reaction mixture was incubated at 37 oC for 16 hours. Then, gDNA was digested with 2.5 uL DNase (Qiagen) in a 100 uL reaction at room temperature for 30 min. RNA was extracted with TRIzol LS Reagent (Invitrogen), and aqueous phase was precipitated with 1 volume of isopropanol and 5 ug Glycogen (Invitrogen) at -80 oC for 1 hour. RNA pellet was collected by centrifugation at 21,000 x g at 4 oC for 1 hour. The pellet was washed with 80% ice-cold ethanol and resuspended in 11.5 uL nuclease-free water. For reverse transcription, RNA was incubated with 0.5 uL 100 uM RT primer p6 (5’-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGNNNNNNNN-3’) and 1 uL 10 mM dNTP at 65 oC for 5 min and cooled on ice. Then, 4 uL 5X RT buffer, 1 uL 100 mM DTT, 1 uL RNaseOUT (40 U/uL) and 1 uL SuperScript IV RT (200 U/uL, Invitrogen) were added and the reaction mixture was incubated at 23 oC for 10 min, 50  oC for 15 min and 80 oC for 10 min. cDNA library was amplified with KAPA2G Robust HotStart polymerase, using primers p7 (5’- GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGAAAGGAAGCCCTGCTTCCTCCAGAGGG-3’, 0.5 uM) and p8 (5’- TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-3’, 0.5 uM). PCR reaction was performed as follows: 95 oC 3 min; 95 oC 15 s, 65 oC 15 s, 72 oC 30 s, 16~18 cycles; and 72 oC 1 min. The PCR product was subjected to double-sided size selection (0.5X, 0.9X) and cleaned up with AMPure XP beads. The resulting product ranged from 200 to 1000 bp. To prepare Illumina sequencing libraries, 5-10 ng PCR product was re-amplified with the Nextera P5 and TruSeq P7 library index primers for 5 cycles. The final PCR product underwent another round of double-sided size selection (0.5X, 0.9X) and cleaned up with AMPure XP beads. The library was sequenced on an Illumina MiSeq in paired-end mode (Read 1: 254 bp; Read2: 55bp).  Amplicon sequencing of synHEK3 reporters: 100~250 ng gDNA or cell lysates were amplified using KAPA2G Robust HotStart ReadyMix with primers p9 (5’-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTCTACCCCGACCACATGAAGCAGC-3’, 0.5 uM) and p10 (5’-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGNNNNNNNNNNGACCATGTCATCGCGCTTCTCGT-3’, 0.5 uM). PCR reaction was performed as follows: 95 oC 3 min; 95 oC 15 s, 68-N oC 15 s, 72 oC 30 s, for 9 cycles (N was cycle number); 95 oC 15 s, 65 oC 15 s, 72 oC 30 s, for 11 cycles; and 72 oC 1 min. To ensure enough coverage and accurate measurement of editing efficiencies, for the K562 synHEK3 pool, we pooled products from at least 16 PCR reactions. The PCR product was purified with AMPure XP beads. 5 ng PCR product was re-amplified with the Nextera P5 and TruSeq P7 library index primers for 5 cycles.   Other amplicon sequencing: Cell lysates were directly used for PCR with the KAPA2G Robust HotStart ReadyMix with primers (0.5 uM each) designed for the endogenous targets. PCR reaction was performed as follows: 95 oC 3 min; 95 oC 15 s, 66-N oC 15 s, 72 oC 40 s, for 8 cycles (N was cycle number); 95 oC 15 s, 60 oC 15 s, 72 oC 40 s, for 11 cycles; and 72 oC 1 min. The PCR product was purified with AMPure XP beads. 5 ng PCR product was re-amplified with the Nextera P5 and TruSeq P7 library index primers for 5 cycles.

treatment_protocol: T7-assisted reporter mapping: We randomly integrated a complex library of the synHEK3 reporters into a monoclonal K562 cell line constitutively expressing the PE2 prime editor. We further bottlenecked the pool to ~500 clones, each containing a unique combination of randomly inserted reporters, and performed T7-assisted reporter mapping on this population.  SynHEK3 amplicon sequencing for prime editing-mediated CTT insertion and Cas9 editing: the K562 synHEK3 pool was transfected with the pU6-Sp-pegRNA-HEK3-insCTT plasmid or Cas9 RNP using the SF Cell Line 4D-Nucleofector X kit L (Lonza).  CRISPRoff experiments: On Day 0, 2 x 106 clone 5 cells were transfected with CRISPRoff-v2.1 (3,000 ng), pU6-Sp-dual-gRNA (1,000 ng) and pmax-GFP (500 ng, Lonza) using the SF Cell Line 4D-Nucleofector X kit L (Lonza). On Day 2, cells were sorted based on high GFP expression (top 20%) on a flow cytometer and expanded. On Day 11, 4 x 105 cells were transfected with the pU6-Sp-pegRNA-HEK3-insCTT plasmid (1,000 ng) using the SF Cell Line 96-well Nucleofector Kit (Lonza). Program code was FF-120.  CRISPRa experiments: On Day 0, 4 x 105 K562 dCas9-VP64 cells were transfected with PB-CMV-MCP-XTEN80-p65-Rta-3xNLS-P2A-T2A-mPlum (600 ng) and paired pU6-Sp-gRNA-2XMS2 (200 ng each) plasmids targeting the same promoter. On Day2, 4 x 105 cells from the previous transfection were transfected with pCMV-PEmax (Addgene: 174820)11 or  PB-PEmax-P2A-mCherry-EF1a-Puro (800 ng) and the pU6-Sp-pegRNA plasmids (400 ng). All transfections were performed using the SF Cell Line 96-well Nucleofector Kit (Lonza) with program FF-120.  For prime editing in shRNA treated cells: we transduced 2 monoclonal line (Clone 3 and Clone 5) individually with lentiviral shRNAs against candidate genes. After 2 days of doxycycline treatment to induce shRNA and GFP expression. For each shRNA, we pooled the 2 clones and transfected the cells with pegRNAs to induce 6-bp insertions. Cells were sorted into GFP positive and negative populations. Amplicon sequencing were performed on the sorted cells.

tag: genotype

value: Wild type

value: CRISPRoff v2, USP7


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_2_P1-12H]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_2_P1-10D]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_2_P1-10F]

description: sci-RNA-seq3 library


title: Prime editing library of syHEK3 reporters in CRISPRoff treated cells [K562-SC1-5-OFF-USP7-Rep1]


title: Prime editing library of syHEK3 reporters in CRISPRoff treated cells [K562-SC1-5-OFF-METTL2A-Rep2]

description:
synHEK3 amplicon sequencing library
CRISPRoff_METTL2A.csv

value: CRISPRoff v2, METTL2A


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_2_P1-10C]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-3_enrich_P1-02F]

description: sci-RNA-seq3 library


title: Prime editing library of syHEK3 reporters in CRISPRoff treated cells [K562-SC1-5-OFF-LRRC8C-Rep2]

description:
synHEK3 amplicon sequencing library
CRISPRoff_LRRC8C.csv

value: CRISPRoff v2, LRRC8C


title: Prime editing library of syHEK3 reporters in CRISPRoff treated cells [K562-SC1-5-OFF-LRRC8C-Rep1]


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_2_P1-09F]

description: sci-RNA-seq3 library


title: Prime editing library of syHEK3 reporters in CRISPRoff treated cells [K562-SC1-5-OFF-Ctrl-Rep2]

description: synHEK3 amplicon sequencing library

value: CRISPRoff v2, Non targeting control


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_2_P1-11B]

description: sci-RNA-seq3 library


title: Prime editing library of syHEK3 reporters in CRISPRoff treated cells [K562-SC1-5-OFF-Ctrl-Rep1]

description: synHEK3 amplicon sequencing library


title: T7-assisted reporter mapping library for synHEK3 reporters in a population derived from 500 clones [T7-500-1_S1_2]

description:
T7-assisted reporter mapping library
T7-500-1_S1_ITR_corrected.txt
T7-500-1_processed.csv

library_source: other

molecule: other


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_2_P1-09D]

description: sci-RNA-seq3 library


title: T7-assisted reporter mapping library for synHEK3 reporters in a population derived from 500 clones [T7-500-1_S1]


title: S87963_S45

city: Baltimore

first: Immunometabolism

last: Department

state: Maryland

address: 1650 Orleans Street

department: Immunometabolism

institute: Johns Hopkins University

data_processing:
The resulting reads were aligned to the human reference genome build hg38 using RSEM with the STAR aligner to obtain posterior mean estimate transcripts per million (pmeTPM) as a measure of gene expression.
Assembly: hg38
Supplementary files format and content: Posterior mean estimate transcripts per million (pmeTPM) as a measure of gene expression

molecule: total RNA

source_name: Patient biopsy

extract_protocol:
Frozen sections were cut onto PEN (polyethylene naphthalate) slides (Leica), stained with hematoxylin and LCM was performed at the SKCCC Core facility using a Leica LMC7000 laser capture microdissection system in the SKCCC Cell Imaging Core. Laser captured tissues from regions highly enriched for tumor cells were collected by manual annotations of regions of interest that outlined tumor nests. RNA was isolated using the ALLPREP RNA/DNA extraction protocol (Qiagen Cat. No. 802804). RNA amounts were measured using a Qubit fluorometer (Invitrogen) and quality were measured using an Agilent Bioanalyzer 2100 with the Pico Chips Kit.
For RNA sequencing of patient biopsy samples, purified RNA was provided to the SKCCC Experimental and Computational Genomics Core to carry out their low-input RNA-seq workflow. Briefly, quality of total RNA was measured by the Agilent Bioanlayzer to determine RNA integrity (RIN). Samples with starting input between 100pg-100ng of total RNA and RIN > 7.0 were considered to have sufficient quality to proceed to construction of whole transcriptome sample-barcoded libraries using the Ovation RNA-Seq System V2 according to the manufacturer’s protocols (Nugen). Quantification of the libraries was performed by qPCR or by the Agilent Bioanalyzer and equimolar concentrations of each library were pooled together, clustered and sequenced on an Illumina Novaseq 6000 platform, with paired end sequencing.

tag: subjectid

tag: batch

tag: change in psa from baseline

tag: psa50 response

value: NO

tag: change in tumor volume from baseline

tag: radiographic response

tag: time point

value: Patient biopsy


title: S87423_S43

value: Pretx


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_2_P1-09C]

description: sci-RNA-seq3 library


title: S87955_S41


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_2_P1-09A]

description: sci-RNA-seq3 library


title: S87421_S40


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_2_P1-09H]

description: sci-RNA-seq3 library


title: S86877_S37

value: YES


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_2_P1-08H]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_2_P1-08F]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_2_P1-08G]

description: sci-RNA-seq3 library


title: S86626_S66


title: S86921_S39


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_2_P1-10A]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_2_P1-10B]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_2_P1-09G]

description: sci-RNA-seq3 library


title: S85293_S38


title: S85059_S70


title: S84972_S67


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_2_P1-09B]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_2_P1-08B]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_2_P1-08C]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_2_P1-08A]

description: sci-RNA-seq3 library


title: S84641_S62


title: S84788_S64


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_2_P1-08E]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_2_P1-08D]

description: sci-RNA-seq3 library


title: S83694_S35


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_2_P1-07F]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_2_P1-07E]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_2_P1-07D]

description: sci-RNA-seq3 library


title: S83540_S34


title: S83329_S33


title: S82796_S56


title: S82780_S53


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_2_P1-07B]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_2_P1-07C]

description: sci-RNA-seq3 library


title: S82600_S58


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_2_P1-09E]

description: sci-RNA-seq3 library


title: S82491_S61


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_2_P1-07A]

description: sci-RNA-seq3 library


title: S82355_S57


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_2_P1-06H]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_2_P1-06G]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_2_P1-07H]

description: sci-RNA-seq3 library


title: S81883_S7


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_2_P1-06F]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_2_P1-06E]

description: sci-RNA-seq3 library


title: S81881_S5


title: Prime editing library of syHEK3 reporters in CRISPRoff treated cells [K562-SC1-5-OFF-METTL2A-Rep1]


title: S81877_S52


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_2_P1-06D]

description: sci-RNA-seq3 library


title: S81301_S2


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_2_P1-06C]

description: sci-RNA-seq3 library


title: S81299_S50


title: S83544_S32


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_2_P1-06A]

description: sci-RNA-seq3 library


title: S81260_S4


title: S81264_S6


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_2_P1-05H]

description: sci-RNA-seq3 library


title: S80847_S1


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_2_P1-05G]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_2_P1-05F]

description: sci-RNA-seq3 library


title: PDT02, Immature ovarian teratomas, case02

city: Chengdu

first: Heng

last: Xu

state: Sichuan

address: 17#, section 3, South Renmin Road

country: China

institute: Sichuan University

description: 10x Genomics

library_source: transcriptomic single cell

data_processing:
The raw data was processed using Cell Ranger (v3.0). Following Cell Ranger processing, a raw unique molecular identifier (UMI) count matrix was generated, which was then converted into a Seurat object by the R package Seurat(v4.0). All Cell Ranger commands were executed with the default settings. Cells with UMI numbers below 500, gene numbers below 300 or greater than 6,000, or mitochondrial-derived UMI counts of more than 15% were considered low-quality and were removed. Furthermore, we used Scrublet (v0.2) to calculate doublet scores and predicted potential doublets for each cell, with expected doublet rate = 0.06. The predicted doublets were then removed. After quality control, 6274, 8435, and 7444 cells were left for each patient for subsequent analysis.
Assembly: GRCh38
Supplementary files format and content: seurat object containing all cells from three PDT samples

source_name: Immature ovarian teratomas

extract_protocol:
Immature ovarian teratomas were dissected and sheared on ice after being washed with Hanks’ balanced salt solution (HBSS). The tissues were then digested for 1 hour at 37°C with collagenase I (2 mg/ ml) (Gibco 1710-0017), collagenase IV (1 mg/ml) (Gibco 1710-4019), and 0.25% pancreatic enzymes (Gibco 25200-056). To remove cell debris and large clumps, the digested tissues were filtered through a 40-mm strainer. At 4°C, the suspensions were pelleted at 500g for 5 minutes. After lysis with 10 RBC Lysis Buffer (Thermo Fisher Scientific, 00-4300-54), the pellets were resuspended in HBSS with 0.04% bovine serum albumin (BSA) to determine cell viability and concentration (Counting Star, Aber Instruments Ltd.). The remaining cells were pelleted at 500g for 5 minutes at 4°C before being stored at 80°C. Finally, single-cell suspensions were diluted to about 5000 cells per milliliter. All tissues were obtained from West China Biobanks at Sichuan University’s West China Hospital’s Department of Clinical Research Management.
According to the directions provided by the manufacturer in the Chromium Single Cell 3’ Reagents Kits v2 User Guide, the Chromium Single Cell 3’ Library & Gel Bead Kit v2 (PN-120237), Chromium Single Cell 3’ Chip Kit v2 (PN-120236), and Chromium i7 Multiplex Kit (PN-120262) were utilized. Phosphate-buffered saline (PBS) + 0.04% BSA was used to wash the single-cell suspension twice. With the use of the TC20 Automated Cell Counter, cell quantity and concentration were verified. A 10x Genomics Chromium Controller machine was used to generate gel beads in emulsion (GEM) from the cells right away. A 10x Genomics Chromium Single Cell 3’ reagent kit (V2 chemistry) was used to prepare barcoded complementary DNAs (cDNAs), which were then recovered, purified, and amplified to provide enough for library construction. With the use of an Agilent Bioanalyzer 2100, library concentration and quality were evaluated. For PE150 sequencing, libraries were performed on Illumina’s NovaSeq 6000 platform.

value: Immature ovarian teratomas


title: PDT03, Immature ovarian teratomas, case03

description: 10x Genomics


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_2_P1-05E]

description: sci-RNA-seq3 library


title: PDT01, Immature ovarian teratomas, case01

description: 10x Genomics


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_2_P1-05D]

description: sci-RNA-seq3 library


title: S82351_S51


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_2_P1-05C]

description: sci-RNA-seq3 library


title: sh 1

city: Changchun

first: Tongjian

last: Zhao

address: 1266 Fujin Road

institute: School of Pharmaceutical Sciences, Jilin University

data_processing:
bcl2fastq,v2.20
fastp,0.22.0
HISAT2,2.2.1
Assembly: hg38
Supplementary files format and content: Comma-separated csv files include fpkm values for each Sample

extract_protocol:
The total RNA of C666-1R-shNC and C666-1R-shHOTAIRM1 was isolated and purified.
Sequencing libraries were generated using NEBNext® Ultra™ RNA Library Prep Kit for Illumina following the manufacturer’s recommendations and index codes were added to attribute sequences to each sample.


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_2_P1-05B]

description: sci-RNA-seq3 library


title: sh 2


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_2_P1-05A]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_2_P1-06B]

description: sci-RNA-seq3 library


title: Control 2

value: NC


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_2_P1-04F]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_2_P1-04E]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_2_P1-04H]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_2_P1-04G]

description: sci-RNA-seq3 library


title: Control 1


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_2_P1-04D]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_2_P1-04B]

description: sci-RNA-seq3 library


title: Hi-C: HeLa WAPL + Sororin depleted G2 rep 3

city: Vienna

first: Paul

last: Batty

address: Zimmermann Platz 10

country: Austria

institute: CCRI

data_processing:
Basecalling software: Illumina RTA
Hi-C samples were preprocessed using a custom nextflow pipeline (https://github.com/gerlichlab/scshic_pipeline). Briefly, bcl2 files were first demultiplexed using bcl2tofastq. Then, fastq files were aligned to hg19 with HeLa SNPs using bwa, aligning read pairs independently. Then, pairsam files were constructed using pairtools (https://github.com/open2c/pairtools), followed by sorting and deduplicating. Then, reads were split into cis sister and trans sister contacts based on the presence of signature mutations using pairtools select: A read was assigned to the Watson strand if it contained two or more A-to-G mutations and no T-to-C mutations. Similarly, if a read contained two or more T-to-C mutations, but no A-to-G mutations it was assigned to the Crick strand. Then, contacts were classified as cis sister contacts if (after correcting for the opposite read-strandedness of Illumina sequencing of the two mates) both mates mapped to the same strand. Conversely, contacts were classified as trans sister contacts if the two mates mapped to opposing strands. Then, pairlib (https://github.com/open2c/pairlib) was used to construct .pairs files.
Assembly: hg19
Supplementary files format and content: Supplementary_files_format_and_content: pairlib derived .pairs files (open2c) containing Hi-C pairs files. Supplementary_files_format_and_content: pairs files were generated using pairlib (https://github.com/open2c/pairlib) using standard parameters. Separate pairs files are provided for cis-sister contacts and trans-sister contacts. Csv files were generated using a custom python script to count different point mutation types.

source_name: WAPL + Sororin depleted G2

growth_protocol: Cell culture for G2 scsHi-C was performed as described in (Mitter et al, 2020, 2022). Wild type medium without selection antibiotics was used to synchronise the cells, and all chemicals used were diluted in this medium to the specified concentration. In brief, asynchronous cells were synchronised to the G1/S boundary with 2 mM thymidine (Sigma Aldrich, T1895) for 16 – 18 h. The following day, cells were washed twice with prewarmed PBS, before the addition of fresh wild type medium. 10-12 h post release, 3 μg mL-1 aphidicolin (Sigma Aldrich, A0781) was added to the cells to arrest them at the G1/S boundary. 2 mM 4-thiothymidine (4sT) (Carbosynth, NT06341) was also supplemented to the medium at this stage. 16 –18 h hours later, cells were washed twice with prewarmed PBS before the addition of 2 mM 4sT. For all G2 samples,10 μM RO-3306 (Sigma Aldrich, SML0569) was added to the cells 5 – 6 h after the second release, and samples were harvested 24 h after the second release. Experiments with protein depletions were performed as follows: For SMC4-AID experiments, 5-Ph-IAA (BioAcademia, 30-003) was added 1 h before the second release to a final concentration of 1 μM. For NIPBL-AID experiments, 5-Ph-IAA was added 8 h after the second release to a final concentration of 1 μM. For WAPL-AID experiments, indole-3-acetic-acid (auxin) (Sigma Aldrich, I5148) was added to a final concentration of 500 μM 14 h after the second release. For all samples where proteins were depleted, auxin or 5-Ph-IAA were added to all wash buffers and medium after they had been added to the samples. For WAPL-AID experiments, siRNA transfections using XWNeg or Sororin siRNAs was performed as described above (section: siRNA transfection), 6-7 h after the release from the thymidine block. To harvest cells, cells were washed twice with PBS, with banging against a solid support each time to remove dead and mitotic cells. Cells were then trypsinised to detach them from the culture flask before quenching with wild type medium. Collected cells were spun down and the pellet washed once with PBS. Cells were spun down, PBS removed, and the cells were fixed with PBS containing 1% methanol-free formaldehyde (Thermo Fisher Scientific, 28906) for 4 min with gentle rotation. Cells were spun down, the formaldehyde removed and 20 mM TRIS-HCl pH 7.5 in PBS added to quench residual formaldehyde. Samples were spun down once more, the liquid removed, and samples stored at -20 °C until further processing. To generate prometaphase samples, cells were treated as described above until the addition of RO-3306. Cells were also cultured in 75 cm2 rather than 25 cm2 flasks to generate sufficient cell numbers for the experiment. RO-3306 was added to a final concentration of 7 μM to cells 7 h after the second release. Before adding RO-3306, cells were washed twice with PBS and banged against a solid support to remove dead and mitotic cells. 14 h after the second release, cells were washed three times with wild type medium. After the final wash, wild type medium containing 200 ng mL-1 nocodazole (Sigma Aldrich, M1404) was added to the cells. 60 minutes later, mitotic cells were detached from the culture flask by banging the flask against a solid support. The medium was then collected, cells spun down and washed once with PBS, before fixation with 1% formaldehyde as described above.

extract_protocol:
Hi-C sample preparation was performed as described in (Mitter et al, 2020, 2022). All centrifugations were performed at 2500 g, apart from those in the ethanol precipitation step, which were performed at 21000 g. Frozen cell pellets were thawed and then lysed using ice cold Hi-C lysis buffer (10 mM TRIS-HCl pH 8 (Sigma), 10 mM NaCl (Sigma), 0.2% Nonidet P-40 substitute (Sigma), 1 × complete EDTA-free protease inhibitor (Roche) for 30 min at 4 °C. The DNA was then digested using Dpn II (375 U DpnII (NEB, R0543 T/M/L) in 1× Dpn II buffer (NEB, B0543S) for 16 h at 37 °C with shaking at 800 rpm on a thermal block. Cells were centrifuged, the supernatant discarded, and fill-in mix added (38 μM biotin-14-dATP (Thermo Fisher Scientific, 19524016), 38 μM dCTP (Thermo Fisher Scientific, R0152), dGTP (Thermo Fisher Scientific, R0162), dTTP (Thermo Fisher Scientific, R0172), 50 U Klenow Polymerase (NEB, M0210L), 1 × NEB 2 buffer, B7002S). Cells were then incubated for 1 h at 37 °C with gentle rotation. Cells were then spun down, the supernatant discarded, ligation mix added (1 × T4 DNA ligase buffer (Thermo Fisher Scientific, B69), 0.1% Triton X-100 (Sigma, X100-100 ML), 100 μg mL-1 BSA (Sigma, A7030-10G), 50 U T4 DNA ligase (Thermo Fisher Scientific, EL0011) before incubation at room temperature for 4 h with gentle rotation. Cells were spun down, the supernatant discarded, and the pellet resuspended in 200 μL PBS. Genomic DNA (gDNA) was then purified using the DNeasy Blood and Tissue Kit (Qiagen, 69506) according to the manufacturer instructions for tissue culture cells. During gDNA purification, after addition of lysis buffer, RNaseA (Qiagen, 19101) and proteinase K (Qiagen, 19131), samples were incubated at 65 °C for 6 h to reverse crosslinks, before continuing with the rest of the protocol.
The purified DNA was then sheared on a Covaris S2 instrument (duty cycle 10%, intensity 5.0, cycles/burst 200) for 25 s. The DNA was sheared in Covaris microTUBEs (Covaris, 520045). Size-selection was then performed to isolate fragments of the correct size using AMPure XP beads (Beckmann Coulter, A63881). A double size selection was performed according to manufacturer instructions. In brief 0.8 x sample volume of beads was first added to the beads, the supernatant was then transferred to a new tube and 0.12 x sample volume beads was then added. The final size-selected DNA was resuspended in dH2O. A biotin-pull down was then performed to select biotin-labelled DNA fragments using Dynabeads MyOne Streptavidin C1 beads (Thermo Fisher Scientific, 65001), resuspended in biotin binding buffer (5 mM TRIS-HCl pH 7.5 (Sigma), 0.5 mM EDTA (AppliChem, 1103.1000), 1M NaCl (Merck). Beads were incubated with the samples for 1 h at room temperature with gentle rotation before two washes with Tween wash buffer (5 mM TRIS-HCl, pH 7.5 (Sigma), 0.5 mM EDTA (Applichem, 1103.1000), 1M NaCl (Merck), 0.05% Tween20 (Sigma, P1379-250ML). A final wash with dH2O was then performed to remove residual detergent before the library preparation. The beads were then resuspended in dH2O, and library prep performed on the beads using the NEBNext Ultra II DNA library prep kit for Illumina (NEB, E7645L), according to the manufacturer instructions. The beads were then washed 4 x with Tween wash buffer. Elution of DNA from the beads was performed by incubation with 95% formamide (Sigma Aldrich, F9037-100ML), 10 mM EDTA (Applichem) at 65 °C for 2 min. Samples were then ethanol precipitated (80% EtOH (Sigma, 32221-1L / 2.5L), 100 mM sodium acetate pH 5.2 (Sigma Aldrich, 71180-1kg), incubation at -70 °C for > 16 h). 1 μL GlycoBlue (Thermo Fisher Scientific, AM9515) was also added during the precipitation to better visualise the pellet. Samples were spun down, the supernatant removed, washed once with 75% EtOH, spun down once more and then resuspended in dH2O. 4sT was then converted to methyl-cytosine using OsO4 / NH4Cl as described in (Mitter et al, 2020, 2022). EtOH precipitation was then performed once more to the converted samples as described above. Finally, qPCR was performed to amplify libraries using the NEBUltra Ultra II DNA library prep kit for Illumina (NEB, E7645L). The final libraries were purified using AMP Pure XP beads (Beckmann Coulter, A63881) at 0.55 x sample volume, according to the manufacturer instructions.

treatment_protocol: HeLa cells were treated with 5-Ph-IAA and drugs to induce G2 or mitotic arrest as specified in the growth protocol.

value: HeLa Kyoto

tag: disease state

value: Cervical carcinoma

value: Sororin depleted by siRNA. WAPL depleted in G2 phase through auxin. Synchronisation to G2 phase with 10 μM RO-3306


title: Hi-C: HeLa WAPL + Sororin depleted G2 rep 4


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_2_P1-04A]

description: sci-RNA-seq3 library


title: Hi-C: HeLa WAPL depleted G2 rep 6

source_name: WAPL depleted G2

value: WAPL depleted in G2 phase through auxin. Synchronisation to G2 phase with 10 μM RO-3306


title: Hi-C: HeLa WAPL + Sororin depleted G2 rep 1


title: Hi-C: HeLa WAPL + Sororin depleted G2 rep 2


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_2_P1-03H]

description: sci-RNA-seq3 library

value: lymphoblast


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_2_P1-03F]

description: sci-RNA-seq3 library

value: lentiviral transduction of a DDR-focused shRNA library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_2_P1-03E]

description: sci-RNA-seq3 library


title: Hi-C: HeLa WAPL depleted G2 rep 5


title: Hi-C: HeLa WAPL depleted G2 rep 4


title: Hi-C: HeLa WAPL depleted G2 rep 3


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_2_P1-03D]

description: sci-RNA-seq3 library


title: Hi-C: HeLa WAPL depleted G2 rep 1


title: Hi-C: HeLa WAPL depleted G2 rep 2


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_2_P1-03B]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_2_P1-03C]

description: sci-RNA-seq3 library


title: Hi-C: HeLa NIPBL depleted G2 rep 10

source_name: NIPBL depleted G2

value: NIPBL depleted in G2 phase through 5-Ph-IAA. Synchronisation to G2 phase with 10 μM RO-3306


title: Hi-C: HeLa NIPBL depleted G2 rep 6


title: Hi-C: HeLa NIPBL depleted G2 rep 8


title: Hi-C: HeLa NIPBL depleted G2 rep 7


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_2_P1-03A]

description: sci-RNA-seq3 library


title: Hi-C: HeLa NIPBL depleted G2 rep 5


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_2_P1-02H]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_2_P1-07G]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_2_P1-02G]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_2_P1-04C]

description: sci-RNA-seq3 library


title: Hi-C: HeLa NIPBL depleted G2 rep 4


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_2_P1-02F]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_2_P1-03G]

description: sci-RNA-seq3 library


title: Hi-C: HeLa NIPBL depleted G2 rep 3


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_2_P1-02E]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_2_P1-02D]

description: sci-RNA-seq3 library


title: Hi-C: HeLa NIPBL depleted G2 rep 2


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_2_P1-02C]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_2_P1-02B]

description: sci-RNA-seq3 library


title: Hi-C: HeLa NIPBL depleted G2 rep 9


title: Hi-C: HeLa SMC4 depleted G2 rep 4

source_name: SMC4 depleted G2

value: SMC4 depleted in G1 phase through 5-Ph-IAA. Synchronisation to G2 phase with 10 μM RO-3306


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_2_P1-02A]

description: sci-RNA-seq3 library


title: Hi-C: HeLa SMC4 depleted G2 rep 3


title: Hi-C: HeLa SMC4 depleted G2 rep 2


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_2_P1-01G]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_2_P1-01F]

description: sci-RNA-seq3 library


title: Hi-C: HeLa NIPBL depleted prometaphase rep 3

source_name: NIPBL depleted prometaphase

value: NIPBL depleted in G2 phase through 5-Ph-IAA. Synchronisation to G2 phase with 7 μM RO-3306, before washout into 200ng/ml nocodazole for 60 min


title: Hi-C: HeLa SMC4 depleted G2 rep 1


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_2_P1-01E]

description: sci-RNA-seq3 library


title: Hi-C: HeLa NIPBL depleted prometaphase rep 2


title: Hi-C: HeLa NIPBL depleted prometaphase rep 1


title: Hi-C: HeLa SMC4 depleted prometaphase rep 1

source_name: SMC4 depleted prometaphase

value: SMC4 depleted in G1 phase through 5-Ph-IAA. Synchronisation to G2 phase with 7 μM RO-3306, before washout into 200ng/ml nocodazole for 60 min


title: Hi-C: HeLa SMC4 depleted prometaphase rep 2


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_2_P1-01C]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_2_P1-01A]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_2_P1-01B]

description: sci-RNA-seq3 library


title: Hi-C: HeLa NIPBL depleted G2 rep 1


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_2_P1-01D]

description: sci-RNA-seq3 library


title: Hi-C: HeLa Wild type prometaphase rep 1

source_name: Wild type prometaphase

value: wild type

value: Synchronisation to G2 phase with 7 μM RO-3306, before washout into 200ng/ml nocodazole for 60 min


title: siTrim-Lac-3

city: NANJING

first: zhang

last: hanqing

address: tong jia xiang 24

institute: china pharmaceutical university

description:
fpkm.anno.xls
total_count.xls
Final quantitative data file.xlsx

data_processing:
The integrity and concentration of RNA were analyzed using Agilent RNA 6000 Nano Kit and Agilent 2100 Bioanalyzer (Agilent Technologies, CA, USA).
Transcriptome sequencing was performed using an Illumina Nova 6000 platform (Illumina Inc., San Diego, CA, USA).
Raw sequenced reads in FASTQ format were filtered to achieve high-quality reads and then mapped to the human genome (GRCh38.100.chr) using HISAT2.
Differentially expressed genes (DEGs) were analyzed using the R package DESeq2 with cutoff of fold change > 1.5 or < 0.67 and p value < 0.05.
Assembly: GRCh38.100.chr
Supplementary files format and content: fpkm.anno.xls file includes FPKM values for each Sample
Supplementary files format and content: total_count.xls file includs count values for each sample

growth_protocol: HCT116 human colorectal carcinoma cells were maintained in MacCoy'5A supplemented with 10% fetal bovine serum (FBS) and antibiotics in humidified atmosphere with 5% CO2 at 37C

extract_protocol:
Total RNA was isolated using TRIzolTM reagent (Thermo Scientific, cat.no.15596026)
RNA-seq was performed by Annoroad Gene Technology (Beijing, China)

treatment_protocol: Approximately 3×105 HCT116 cells were seeded into six-well plates for 24 h and then transfected with 10 nM siRNA using 5 μL lipofectamineTM RNAiMAX reagent for 24h, followed by treatment with 10 mM sodium lactate or the matching solvent and cultured for another 24 h.

value: Human colorectal carcinoma cell

value: Trim28 silence

value: sodium lactate


title: siTrim-Lac-2


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_P1-12G]

description: sci-RNA-seq3 library


title: siTrim-Lac-4


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_P1-12F]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_P1-12D]

description: sci-RNA-seq3 library


title: siTrim-Con-4

value: solvent


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_2_P1-01H]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_P1-12C]

description: sci-RNA-seq3 library


title: siTrim-Con-3


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_P1-12B]

description: sci-RNA-seq3 library


title: siTrim-Con-2


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_P1-12A]

description: sci-RNA-seq3 library


title: siTrim-Con-1


title: siCtrl-Lac-4

value: Control silence


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_P1-11F]

description: sci-RNA-seq3 library


title: siCtrl-Lac-3


title: siCtrl-Lac-2


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_P1-11G]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_P1-12H]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_P1-11D]

description: sci-RNA-seq3 library


title: Hi-C: HeLa Wild type prometaphase rep 2

value: HeLa Kyoto


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_P1-11H]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_P1-12E]

description: sci-RNA-seq3 library


title: siCtrl-Lac-1


title: siCtrl-Con-4


title: siCtrl-Con-3


title: siTrim-Lac-1


title: siCtrl-Con-1


title: siCtrl-Con-2


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_P1-11B]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_P1-11A]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_P1-10H]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_P1-10G]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_P1-10E]

description: sci-RNA-seq3 library


title: NSCs of SVZ_4

city: Guangzhou

first: Yi

last: Liu

address: Guangzhou Dadao Bei Street 1838

institute: Nanfang Hospital of Southern Medical University

data_processing:
Sequencing libraries were generated using NEB Next® Ultra RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer’s recommendations, and index codes were added to attribute sequences to each sample
The clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumina) according to the manufacturer’s instructions. After cluster generation, the library preparations were sequenced on an Illumina Novaseq platform and 150 bp paired-end reads were generated
Feature Counts v1.5.0-p3 was used to count the reads numbers mapped to each gene. Next, FPKM of each gene was calculated based on the length of the gene and reads count mapped to this gene
Assembly: Hisat2 v2.0.5
Supplementary files format and content: tab-delimited text files include FPKM values for each Sample

source_name: Ctrl

growth_protocol: For NSCs senescence sequencing, CD133+ NSCs from the 3VF adjacent to ACP (n=7) and from the relatively normal SVZ far away from frontal glioma (n=4) were prepared with MACS by using the Indirect CD133 MicroBead Kit (Miltenyi Biotec)

extract_protocol:
A total amount of 1 μg RNA per sample was used as input material for RNA sample preparations
Sequencing libraries were generated using NEB Next® Ultra RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer’s recommendations

value: Brain

tag: group

value: NSCs from SVZ

value: neural stem cells


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_P1-10F]

description: sci-RNA-seq3 library

value: lymphoblast


title: NSCs of SVZ_3


title: NSCs of SVZ_2


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_P1-11E]

description: sci-RNA-seq3 library

value: lentiviral transduction of a DDR-focused shRNA library


title: htNSCs_7

source_name: Experiment

value: htNSCs from 3VF adjacent to ACP


title: NSCs of SVZ_1


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_P1-10D]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_P1-11C]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_P1-10C]

description: sci-RNA-seq3 library


title: htNSCs_6


title: htNSCs_5


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_P1-10B]

description: sci-RNA-seq3 library


title: htNSCs_4


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_P1-09H]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_P1-09G]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_P1-10A]

description: sci-RNA-seq3 library


title: htNSCs_3


title: htNSCs_2


title: htNSCs_1


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_P1-09F]

description: sci-RNA-seq3 library


title: PCP_4

source_name: Brain tumor tissue

growth_protocol: To test whether high content of lipids was originated from ACP cells, the ACP tissues (n=5), PCP tissues (n=4) and PA (n=3) tissues were prepared and sequenced.

value: Brain tumor tissue

value: tumor tissue

value: PCP


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_P1-09E]

description: sci-RNA-seq3 library


title: PCP_3


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_P1-09D]

description: sci-RNA-seq3 library


title: PCP_2


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_P1-09A]

description: sci-RNA-seq3 library


title: PCP_1


title: PA_3

value: NFPA


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_P1-09C]

description: sci-RNA-seq3 library


title: PA_2


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_P1-08H]

description: sci-RNA-seq3 library


title: PA_1


title: ACP_5

value: ACP


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_P1-08E]

description: sci-RNA-seq3 library


title: ACP_3


title: ACP_4


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_P1-08C]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_P1-08F]

description: sci-RNA-seq3 library


title: ACP_1


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_P1-07H]

description: sci-RNA-seq3 library


title: ACP_2


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_P1-08A]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_P1-08B]

description: sci-RNA-seq3 library


title: RNA-Seq of AML patient (UD5), relapse phase, replicate 3

city: Firenze

first: Gianluca

last: Mattei

state: FI

address: Via di S. Marta, 3

country: Italy

institute: Università degli Studi di Firenze

data_processing:
DNA from each of three pairs of matched AMLs was sequenced on the GridION X5 instrument with a  run-time of 48h by using five or six individual R9.4 flow
The reads generated by each run were base-called using Guppy v2.0.2
The and quality assessment was performed with FastQC
To perform counts of transcripts from paired-end reads we used Salmon
DESeq2 was used to perform counts normalization and the statistical analysis
Assembly: hg19
Supplementary files format and content: Processed data files contain genes on each row and counts for each sample in the column

source_name: bone marrow

extract_protocol:
RNA was extracted from cryopreserved bone marrow (BM) mononuclear cells using QIAGEN’s AllPrep DNA/RNA kit.
RNAseq libraries were prepared using TruSeq Stranded Total RNA Library Prep Gold

tag: phase

value: relapse

value: AML

value: bone marrow


title: RNA-Seq of AML patient (UD10), onset phase, replicate 3

value: onset


title: RNA-Seq of AML patient (UD10), relapse phase, replicate 3


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_P1-07F]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_P1-07G]

description: sci-RNA-seq3 library


title: RNA-Seq of AML patient (AML2), onset phase, replicate 3


title: RNA-Seq of AML patient (AML2), relapse phase, replicate 3


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_P1-07E]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_P1-07D]

description: sci-RNA-seq3 library


title: RNA-Seq of AML patient (UD5), onset phase, replicate 2


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_P1-07C]

description: sci-RNA-seq3 library


title: RNA-Seq of AML patient (UD5), relapse phase, replicate 2


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_P1-08G]

description: sci-RNA-seq3 library


title: RNA-Seq of AML patient (UD10), onset phase, replicate 2


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_P1-08D]

description: sci-RNA-seq3 library


title: RNA-Seq of AML patient (UD10), relapse phase, replicate 2


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_P1-07A]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_P1-06H]

description: sci-RNA-seq3 library


title: RNA-Seq of AML patient (AML2), onset phase, replicate 2


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_P1-06G]

description: sci-RNA-seq3 library


title: RNA-Seq of AML patient (AML2), relapse phase, replicate 2


title: RNA-Seq of AML patient (UD5), onset phase, replicate 1


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_P1-06F]

description: sci-RNA-seq3 library


title: RNA-Seq of AML patient (UD5), relapse phase, replicate 1


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_P1-06E]

description: sci-RNA-seq3 library


title: RNA-Seq of AML patient (UD10), onset phase, replicate 1


title: RNA-Seq of AML patient (AML2), onset phase, replicate 1


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_P1-06A]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_P1-06D]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_P1-06C]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_P1-06B]

description: sci-RNA-seq3 library


title: RNA-Seq of AML patient (AML2), relapse phase, replicate 1


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_P1-07B]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_P1-05E]

description: sci-RNA-seq3 library


title: Induced Treg cells, STM2457_2, Day5

city: Changsha

first: Shuang

last: Lu

state: Hunan

address: No.139 Renmin Middle Road

institute: The 2nd Xiangya Hospital

data_processing:
Illumina Casava2.19 software used for basecalling.
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence, then mapped to hg38 whole genome using hisat2 v2.0.5 with parameters hisat2 [options]* -x <hisat2-idx> {-1 <m1> -2 <m2> | -U <r> | --sra-acc <SRA accession number>} [-S <hit>]
Fragments Per Kilobase of exon per Megabase of library size (FPKM) were calculated using a protocol from Trapnell,Cole et al., Transcript assembly and quantification by RNA-seq reveals unannotated transcripts and isoform switching during cell differentiation. Nat. Biotechnol, 2010. In short, exons from all isoforms of a gene were merged to create one meta-transcript. The number of reads falling in the exons of this meta-transcript were counted and normalized by the size of the meta-transcript and by the size of the library.
Read count extraction and normalization were performed using CLC genomic benchwork
Assembly: hg38
Supplementary files format and content: tab-delimited text files include RPKM values for each Sample

source_name: Induced Treg cells

growth_protocol: Induced human Treg cells were maintained in RPMI 1640 supplemented with 10% fetal bovine serum (FBS) and antibiotics in humidified atmosphere with 5% CO2 at 37C

extract_protocol:
Retinas were removed, flash frozen on dry ice, and RNA was harvested using Trizol reagent. Illumina TruSeq RNA Sample Prep Kit (Cat#FC-122-1001) was used with 1 ug of total RNA for the construction of sequencing libraries.
RNA libraries were prepared for sequencing using standard Illumina protocols.

treatment_protocol: In vitro differentiation was induced by treatment of anti-CD3 antibodies (2μg/ml, Sigma-Aldrich) and anti-CD28 (1μg/ml, Sigma-Aldrich) antibodies, IL-2 (5ng/ml, Peprotech), and TGF-β (5ng/ml, R&D System) for 5 days.

value: Induced Treg cells

value: T cells

value: induced Treg differentiation

value: Day 5


title: Induced Treg cells, STM2457_4, Day5


title: Induced Treg cells, STM2457_1, Day5


title: Induced Treg cells, DMSO_4, Day5

value: DMSO


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_P1-05C]

description: sci-RNA-seq3 library

value: lymphoblast


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_P1-05D]

description: sci-RNA-seq3 library

value: lentiviral transduction of a DDR-focused shRNA library


title: Induced Treg cells, DMSO_2, Day5


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_P1-05A]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_P1-05B]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_P1-04H]

description: sci-RNA-seq3 library


title: Induced Treg cells, DMSO_1, Day5


title: RNA-Seq of AML patient (UD10), relapse phase, replicate 1


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_P1-04G]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_P1-09B]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_P1-05H]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_P1-05G]

description: sci-RNA-seq3 library


title: Induced Treg cells, STM2457_3, Day5


title: CB5

city: La Jolla

first: Judith

last: Varner

state: CA

address: 3855 Health Sciences Dr.

department: Moores Cancer Center

institute: University of California, San Diego

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 COVI19 Patient 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

tag: tissue from postmortem or living patient

value: Living


title: CB6

tag: tissue


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_P1-05F]

description: sci-RNA-seq3 library

tag: cell line

tag: cell type


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_P1-04F]

description: sci-RNA-seq3 library

tag: treatment


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_P1-04C]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_P1-04D]

description: sci-RNA-seq3 library


title: Cb4


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_P1-04E]

description: sci-RNA-seq3 library


title: CB3


title: Induced Treg cells, DMSO_3, Day5

tag: genotype


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_P1-04B]

description: sci-RNA-seq3 library


title: Ischemia+Eugenol Treatment 1

city: Orlando

first: manish

middle: Kumar

last: Gupta

state: Florida

address: 6900 Lake Nona Blvd

department: Burnett School of Biomedical Sciences

institute: University of Central Florida

data_processing:
Filtered and sequenced reads were mapped using HISAT2 algorithms with reference genome Rattus norvegicus release 97
Transcripts were assembled using StringTie v1.3.3b
CuffCompare v2.1.1 was used to compare transcripts
Fragments per kilobase of transcript sequence per Millions base pairs sequenced (FPKM), the most common method were used in estimating gene expression levels
Read count was adjusted by Trimmed Mean of M-values (TMM), then differential expression analysis was performed by using the EdgeR R package
Assembly: Rattus norvegicus release 97
Supplementary files format and content: Differentially regulated genes for NRVC treated with ARVs were identified using DESeq2 R package

organism: Rattus norvegicus

source_name: Primary Cardiomyocytes

growth_protocol: Neonatal rat ventricular cardiomyocytes (NRVCs) were isolated from the 2-3 days old Harlan Sprague–Dawley rats and plated in 6 cm disk at 1x106 cells per well.

extract_protocol:
Total RNA was isolated using mini spin column from Qiagen
Libraries were prepared according to Illumina's instructions In brief, first and second strand synthesis was performed using standard protocol of TruSeq RNA Sample Preparation Kit (Illumina, San Diego, CA). cDNA fragments ~400 bp were obtained on gel electrophoresis and followed by PCR amplification. cDNA library was sequenced using Illumina’s HiSeq 2000 with four RNA-seq libraries per lane.

treatment_protocol: NRVCs were treated under normoxic and ischemic condition (1% O2, 5% CO2) with 10µM Eugenol (Enzo Life Sciences Farmingdale, New York) for 24 hours

tag: strain

value: Sprague Dawley

tag: cells isolated

value: Heart Ventricles

value: Ischemia+Eugenol Treatment

tag: duration of treatment

value: 24 hours


title: CB8


title: Ischemia+Eugenol Treatment 2


title: Ischemia+DMSO Treatment 2

value: Ischemia+DMSO Treatment


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_P1-04A]

description: sci-RNA-seq3 library


title: CB7


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_P1-03H]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_P1-03G]

description: sci-RNA-seq3 library


title: Normoxia+EugenolTreatment 2

value: Normoxia+Eugenol Treatment


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_P1-03F]

description: sci-RNA-seq3 library


title: Normoxia+DMSO Treatment 2

value: Normoxia+DMSO Treatment


title: Normoxia+EugenolTreatment 1

value: Normoxia+EugenolTreatment


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_P1-03C]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_P1-03B]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_P1-03D]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_P1-03E]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_P1-03A]

description: sci-RNA-seq3 library


title: Normoxia+DMSO Treatment 1


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_P1-02G]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_P1-02E]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_P1-02F]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_P1-02H]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_P1-02D]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_P1-02C]

description: sci-RNA-seq3 library


title: Ischemia+DMSO Treatment 1


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_P1-02A]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_P1-02B]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_P1-01G]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_P1-01F]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_P1-01H]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_P1-01E]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_P1-01C]

description: sci-RNA-seq3 library


title: VMH-WT-HFD-2

type: RNA

city: Ciudad de México

first: Ricardo

last: Orozco-Solis

state: TLALPAN

address: Periférico Sur 4809, Arenal Tepepan

country: Mexico

institute: Instituto Nacional de Medicina Genomica

description: VMH of control mice, high fat diet

hyb_protocol: 100 ng (VMH) or 150 ng (BAT) of total RNA per sample was used as a template to obtain cDNA with the GeneChip cDNA synthesis Kit (Affymetrix, Santa Clara, CA). Mouse Gene ST arrays 2.0 (Affymetrix) were used to determine the transcriptome expression levels in the three groups of mice.

scan_protocol: The arrays were scanned in the GeneChip Scanner 3000 7G (Affymetrix) and the Command Console Software was used to produce the .CEL intensity file.

data_processing: Data were analyzed in Affymetrix Expression Console software v1.1.1 using the PLIER algorithm to generate probe level summarization files (*.CHP). (Algorithm: PLIER v 2.0; Quantification Scale: Linear; Quantification Type: Signal and Detection P-Value; Background: PM-GCBG; Normalization Method: Sketch-Quantile).

label: TdT

organism: Mus musculus

source_name: VMH of control mice, high fat diet

label_protocol: The fragmented single-stranded DNA is labeled with recombinant terminal deoxynucleotidyl transferase (TdT) and the Affymetrix proprietary DNA Labeling Reagent that is covalently linked to biotin

extract_protocol: Brain and BAT were rapidly removed after sacrifice and frozen in dry ice and stored at -80°C. Brains were sectioned in a cryostat at 100 um and the VMH dissected. Total RNA extraction of VMH and 20 mg of BAT was carriedout using Trizol

treatment_protocol: Control and Gfap-Bmal1 cKO mice were fed either a standard or high fat diet for 16 weeks prior to tissue harvest

value: control

tag: diet

value: high fat diet

value: Ventromedial Hypothalamus (VMH)


title: VMH-KO-HFD-2

description: VMH of Gfap-Bmal1 cKO mice, high fat diet

value: Gfap-Bmal1 cKO


title: VMH-WT-HFD-3

description: VMH of control mice, high fat diet


title: VMH-KO-HFD-3

source_name: VMH of Gfap-Bmal1 cKO mice, high fat diet


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_P1-01B]

description: sci-RNA-seq3 library

value: lymphoblast


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_P1-01A]

description: sci-RNA-seq3 library

value: lentiviral transduction of a DDR-focused shRNA library


title: VMH-KO-SD-1

description: VMH of Gfap-Bmal1 cKO mice, standard diet

value: standard diet


title: VMH-KO-SD-3

source_name: VMH of Gfap-Bmal1 cKO mice, standard diet


title: VMH-WT-SD-3

description: VMH of control mice, standard diet

source_name: VMH of control mice, standard diet


title: VMH-KO-HFD-1


title: VMH-WT-HFD-1

description: VMH of control mice, high fat diet


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-2_enrich_P1-01D]

description: sci-RNA-seq3 library


title: VMH-KO-SD-2


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

description: sci-RNA-seq3 library


title: BAT-KO-HFD-1

description: BAT of Gfap-Bmal1 cKO mice, high fat diet

value: Brown Adipose Tissue (BAT)


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

description: sci-RNA-seq3 library


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

description: sci-RNA-seq3 library


title: BAT-WT-HFD-1

description: BAT of control mice, high fat diet

source_name: BAT of control mice, high fat diet


title: BAT-WT-HFD-3

description: BAT of control mice, high fat diet


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

description: sci-RNA-seq3 library


title: BAT-WT-HFD-2

description: BAT of control mice, high fat diet


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

description: sci-RNA-seq3 library


title: BAT-KO-HFD-2

source_name: BAT of Gfap-Bmal1 cKO mice, high fat diet


title: VMH-WT-SD-1

description: VMH of control mice, standard diet


title: VMH-WT-SD-2

description: VMH of control mice, standard diet


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

description: sci-RNA-seq3 library


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

description: sci-RNA-seq3 library


title: CB2

value: bronchoalveolar lavage

value: FFPE and sectioned cell pellets

value: Living


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_2_P1-11E]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_2_P1-11H]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_2_P1-11G]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_2_P1-11F]

description: sci-RNA-seq3 library


title: CB1


title: BAT-KO-HFD-3


title: BAT-KO-SD-2

description: BAT of Gfap-Bmal1 cKO mice, standard diet


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_2_P1-11D]

description: sci-RNA-seq3 library


title: BAT-KO-SD-3

source_name: BAT of Gfap-Bmal1 cKO mice, standard diet


title: BAT-KO-SD-1


title: NB3

source_name: FFPE Normal human brochoalveolar lavage cells

value: Normal


title: NB4


title: NB5


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_2_P1-11C]

description: sci-RNA-seq3 library


title: NB2


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_2_P1-10H]

description: sci-RNA-seq3 library


title: C14

source_name: FFPE COVID19 patient lung

value: lung

value: FFPE tissue section

value: Postmortem


title: C13


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_2_P1-10G]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_2_P1-10F]

description: sci-RNA-seq3 library


title: C12


title: C11


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_2_P1-10E]

description: sci-RNA-seq3 library


title: BAT-WT-SD-1

description: BAT of control mice, standard diet

source_name: BAT of control mice, standard diet


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_2_P1-10C]

description: sci-RNA-seq3 library


title: BAT-WT-SD-2

description: BAT of control mice, standard diet


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_2_P1-11B]

description: sci-RNA-seq3 library


title: C7


title: C6


title: BAT-WT-SD-3

description: BAT of control mice, standard diet


title: C5


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

description: sci-RNA-seq3 library


title: C4


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_2_P1-11A]

description: sci-RNA-seq3 library


title: C3


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_2_P1-10A]

description: sci-RNA-seq3 library


title: C8


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

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_2_P1-10D]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-4_enrich_2_P1-10B]

description: sci-RNA-seq3 library


title: C1


title: N5

source_name: FFPE Normal human lung


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

description: sci-RNA-seq3 library


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

description: sci-RNA-seq3 library


title: N4


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

description: sci-RNA-seq3 library


title: N3


title: N2


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

description: sci-RNA-seq3 library


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

description: sci-RNA-seq3 library


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

description: sci-RNA-seq3 library


title: N1


title: PanCK DSP-1001660005232-A-H12

description:
PanCK
RNA (amplified oligo nucleotide probes)

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

source_name: C1 Covid 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: C2


title: PanCK DSP-1001660005232-A-H11


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

description: sci-RNA-seq3 library


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

description: sci-RNA-seq3 library


title: PanCK DSP-1001660005232-A-H10


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

description: sci-RNA-seq3 library


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

description: sci-RNA-seq3 library


title: PanCK DSP-1001660005232-A-H09

source_name: Normal Lung


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

description: sci-RNA-seq3 library


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

description: sci-RNA-seq3 library


title: PanCK DSP-1001660005232-A-H08


title: PanCK DSP-1001660005232-A-H05


title: PanCK DSP-1001660005232-A-H07


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

description: sci-RNA-seq3 library