organism: Homo sapiens
attributes.tag: library_ID
attributes.value: HEI193_0_GEX_3_S3
attributes.tag: isolate
attributes.value: schwannoma
attributes.tag: age
attributes.value: 56
attributes.tag: sex
attributes.value: male
attributes.tag: biomaterial_provider
attributes.value: Raleigh Lab UCSF Helen Diller Family Comprehensive Cancer Center 1450 3rd Street, San Francisco 94203
attributes.tag: tissue
attributes.value: cells
attributes.tag: platform
attributes.value: ILLUMINA
attributes.tag: instrument_model
attributes.value: Illumina NovaSeq 6000
attributes.tag: design_description
attributes.value: sgRNA library was packaged into lentivirus using HEK-293T cells. HEI-193 cultures were transduced to an MOI of 0.1 and FACS sorted for sgRNA+ cells after 48 hours. Radiotherapy was then delivered to either 0Gy or 1.8Gy x 5 daily fractions using the X-Rad 320 irradiator (Precision X-Ray). Following completion of radiotherapy, cells were harvested with Trypsin and prepared following the Nuclei Isolation for Single Cell Multiome ATAC + Gene Expression Sequencing 10x Protocol (10x Genomics, CG000365 Rev C). Briefly, 1-2 105 cells were incubated in chilled Lysis Buffer (Tris-HCl (pH 7.4) 10 mM, NaCl 10 mM, MgCl2 3 mM, Tween-20 0.1%, Nonidet P40 Substitute 0.1%, Digitonin 0.01%, BSA 1%, DTT 1mM, RNase inhibitor 1 U/µL) on ice for 4 minutes, washed with Wash Buffer (Tris-HCl (pH 7.4) 10 mM, NaCl 10 mM, MgCl2 3 mM, Tween-20 0.1%, BSA 1%, DTT 1 mM, RNase inhibitor 1 U/uL) 3 times, and resuspended in diluted nuclei buffer. Nuclei were counted and membrane integrity was evaluated by Trypan staining on the Countess II FL Automated Cell Counter (Thermo). Nuclei suspensions were diluted to approximately 3,000 nuclei/µL and processed according to the 10x Chromium Next GEM Single Cell Multiome ATAC + Gene Expression protocol (CG000338 Rev A). To recover sgRNA identities from single-nuclei RNA fractions, CROP-seq guides were amplified into dual-indexed Illumina libraries from the cDNA product of the 10x multiome protocol as described above with a three-round hemi-nested PCR as previously described85. 15 ng of full-length cDNA product was amplified with primers binding to the sgRNA constant region and 10x Genomics Read 1 Adaptor. Two subsequent PCR steps were performed to introduce i5 and i7 indices and Illumina P5 and P7 adaptors. After the first round of PCR, each product was size-selected using SPRI beads at 1.0x. Subsequent PCRs were conducted with 1 ng product. The final PCR product was size selected with 0.5x and then 1.0x SPRI beads and library quality was assessed by TapeStation high-sensitivity D1000 analysis (Thermo). CROP-seq libraries were pooled with gene expression libraries and sequenced on an Illumina NovaSeq 6000 using the paired end 100 bp protocol.
attributes.tag: filetype
attributes.value: fastq
attributes.tag: assembly
attributes.value: GRCh38
attributes.tag: filename
attributes.value: HEI193_0_GEX_3_S3_L001_R1_001.fastq.gz
attributes.tag: filename2
attributes.value: HEI193_0_GEX_3_S3_L002_R1_001.fastq.gz
attributes.tag: filename3
attributes.value: HEI193_0_GEX_3_S3_L003_R1_001.fastq.gz
attributes.tag: filename4
attributes.value: HEI193_0_GEX_3_S3_L004_R1_001.fastq.gz
attributes.tag: filename5
attributes.value: HEI193_0_GEX_3_S3_L001_R2_001.fastq.gz
attributes.tag: filename6
attributes.value: HEI193_0_GEX_3_S3_L002_R2_001.fastq.gz
attributes.tag: filename7
attributes.value: HEI193_0_GEX_3_S3_L003_R2_001.fastq.gz
attributes.tag: filename8
attributes.value: HEI193_0_GEX_3_S3_L004_R2_001.fastq.gz
attributes.tag: filename9
attributes.value: HEI193_0_GEX_3_S3_L001_I1_001.fastq.gz
attributes.tag: filename10
attributes.value: HEI193_0_GEX_3_S3_L002_I1_001.fastq.gz
attributes.tag: filename11
attributes.value: HEI193_0_GEX_3_S3_L003_I1_001.fastq.gz
attributes.tag: filename12
attributes.value: HEI193_0_GEX_3_S3_L004_I1_001.fastq.gz
attributes.tag: filename13
attributes.value: HEI193_0_GEX_3_S3_L001_I2_001.fastq.gz
attributes.tag: filename14
attributes.value: HEI193_0_GEX_3_S3_L002_I2_001.fastq.gz
attributes.tag: filename15
attributes.value: HEI193_0_GEX_3_S3_L003_I2_001.fastq.gz
attributes.tag: filename16
attributes.value: HEI193_0_GEX_3_S3_L004_I2_001.fastq.gz
attributes.tag: BioSampleModel
attributes.value: Human