GEO Logo
   NCBI > GEO > Accession DisplayHelp Not logged in | LoginHelp
GEO help: Mouse over screen elements for information.
Sample GSM4471660 Query DataSets for GSM4471660
Status Public on Jul 23, 2020
Title Mouse-Cortex_HyPBase_scCC
Sample type SRA
Source name Mouse cortex
Organism Mus musculus
Characteristics tissue: cerebral cortex
strain: C57BL6/J
Treatment protocol We mixed equal volumes of each virus and performed intracranial cortical injections of the mixture into newborn wild-type C57BL/6J pups (P0-2). As a gating control, we injected one litter-matched animal with AAV9-PB-SRT-tdTomato only. After 2 to 4 weeks, we sacrificed mice and dissected the cortex (8 libraries) or hippocampus (1 library).
Growth protocol All mouse experiments were done following procedures described in (Cammack et al., 2019). In brief, we cloned the PB-SRT-tdTomato and HyPBase constructs into AAV vectors. The Hope Center Viral Vectors Core at Washington University in St. Louis packaged each construct in AAV9 capsids. Titers for each virus ranged between 1.1e13 and 2.2e13 viral genomes/ml. All animal practices and procedures were approved by the Washington University in St. Louis Institutional Animal Care and Use Committee (IACUC) in accordance with National Institutes of Health (NIH) guidelines.
Extracted molecule polyA RNA
Extraction protocol Mouse cortical tissues were dissociated to single suspensions following a modification of previously published methods (Avey et al., 2018; Saxena et al., 2012). We incubated samples in a papain solution containing Hibernate-A with 5% v/v trehalose, 1x B-27 Supplement, 0.7 mM EDTA, 70 µM 2-mercaptoethanol, and 2.8 mg/ml papain. After incubation at 37ºC, cells were treated with DNaseI, triturated through increasingly narrow fire-polished pipettes, and passed through a 40-micron filter prewetted with resuspension solution: Hibernate-A containing 5% v/v trehalose, 0.5% Ovomucoid Trypsin Inhibitor, 0.5% Bovine Serum Albumin (BSA), 33 µg/ml DNaseI (Worthington), and 1x B-27 Supplement. The filter was washed with 6 ml of resuspension solution. The resulting suspension was centrifuged for 4 minutes at 250 g. The supernatant was discarded. The pellet was then resuspended in 2 ml of resuspension solution and resuspended by gentle pipetting. We eliminated subcellular debris using gradient centrifugation. We first prepared a working solution of 30% w/v OptiPrep Density Gradient Medium mixed with an equal volume of 1x Hank’s Balanced Salt Solution (HBSS) with 0.5% BSA. We then prepared solutions of densities 1.057, 1.043, 1.036, and 1.029 g/ml using by combining the working solution with resuspension solution at ratios of 0.33:0.67, 0.23:0.77, 0.18:0.82, and 0.13:0.87, respectively. We layered 1 ml aliquots of each solution in a 15 ml conical tube beginning with the densest solution on the bottom. The cell suspension was added last to the tube and centrifuged for 20 minutes at 800g at 12ºC. The top layer was then aspirated and purified cells were isolated from the remaining layers. These cells were then resuspended in FACS buffer: 1x HBSS, 2 mM MgCl2, 2 mM MgSO4, 1.25 mM CaCl2, 1 mM D-glucose, 0.02% BSA, and 5% v/v trehalose. Cells were centrifuged for 4 minutes at 250 g, the supernatant was discarded, and the pellet was resuspended in FACS buffer by gentle pipetting. Cells were then sorted based on fluorescence activity. As a gating control, we analyzed cells from cortices injected with AAV9-PB-SRT-tdTomato only. We then collected cells from brains transfected with AAV9-PB-SRT-tdTomato and AAV9-HyPBase whose fluorescence values exceeded the gate. After sorting, cells were centrifuged for 3 minutes at 250 g. The supernatant was discarded and cells were resuspended in FACS buffer at a concentration appropriate for 10x Chromium 3’ scRNA-seq library preparation. Single cell RNA-seq libraries were prepared using 10x Genomics’ Chromium Single Cell 3’ Library and Gel Bead Kit. Each replicate was targeted for recovery of 6,000 cells. Library preparation followed a modified version of the manufacturer’s protocol. We prepared the Single Cell Master Mix without RT Primer, replacing it with an equivalent volume of Low TE Buffer. Gel-in-emulsion (GEM) generation and GEM-RT incubation proceeded as instructed. At the end of Post GEM-RT cleanup, we added 36.5 µl Elution Solution I and transferred 36 µl of the eluted sample to a new tube (instead of 35.5 µl and 35 µl, respectively). The eluate was split into two 18 µl aliquots and kept at –20ºC until ready for further processing. One fraction was kept for single cell calling cards library preparation, while the other half was further processed into a single cell RNA-seq library.
To amplify self-reporting transcripts from single cell RNA-seq libraries, we took 9 µl of RT product (the other half was kept in reserve) and added it to 25 µl Kapa HiFi HotStart ReadyMix and 15 µl ddH2O. We then prepared a PCR primer cocktail comprising 5 µl of 100 µM Bio_Illumina_Seq1_scCC_10X_3xPT primer, 5 µl of 100 µM Bio_Long_PB_LTR_3xPT, and 10 µl of 10 mM Tris-HCl, 0.1 mM EDTA buffer. One µl of this cocktail was added to the PCR mixture and placed in a thermocycler. Thermocycling settings were as follows: 98ºC for 3 minutes; 20-22 cycles of 98ºC for 20 seconds–67ºC for 30 seconds–72ºC for 5 minutes; 72ºC for 10 minutes; 4ºC forever. PCR purification was performed with 30 µl AMPure XP beads (0.6x ratio) as described previously. The resulting library was quantitated on an Agilent TapeStation 4200 System using the High Sensitivity D5000 ScreenTape. Single cell calling card library preparation was performed using the Nextera Mate Pair Sample Prep Kit with modifications to the manufacturer’s protocol. The library was circularized by bringing 300 fmol (approximately 200 ng) of DNA up to a final volume of 268 µl with ddH2O, then adding 30 µl Circularization Buffer 10x and 2 µl Circularization Ligase (final concentration: 1 nM). This reaction was incubated overnight (12-16 hours) at 30ºC. After removal of linear DNA (following manufacturer’s instructions), we sheared the library on a Covaris E220 Focused-ultrasonicator with the following settings–peak power intensity: 200; duty factor: 20%; cycles per burst: 200; time: 40 seconds; temperature: 6ºC. The library preparation was performed per manufacturer’s instructions until adapter ligation. We designed custom adapters (Table S5) so that the standard Illumina sequencing primers would not interfere with our library. Adapters were prepared by combining 4.5 µl of 100 µM scCC_P5_adapter, 4.5 µl of 100 µM scCC_P7_adapter, and 1 µl of NEBuffer 2, then heating in a thermocycler at 95ºC for 5 minutes, then holding at 70ºC for 15 minutes, then ramping down at 1% until it reached 25ºC, holding at that temperature for 5 minutes, before keeping at 4ºC forever. One microliter of this custom adapter mix was used in place of the manufacturer’s recommended DNA Adapter Index. The ligation product was cleaned per manufacturer’s instructions. For the final PCR, the master mix was created by combining 20 µl Enhanced PCR Mix with 28 µl of ddH2O and 1 µl each of 25 µM scCC_P5_primer and 25 µM scCC_P7_primer. This was then added to the streptavidin bead-bound DNA and amplified under the following conditions: 98ºC for 30 seconds; 15 cycles of: 98ºC for 10 seconds–60ºC for 30 seconds–72ºC for 2 minutes; 72ºC for 5 minutes; 4ºC forever. All of the PCR supernatant was transferred to a new tube and purified with 35 µl (0.7x) AMPure XP beads following manufacturer’s instructions. The final library was eluted in 25 µl Elution Buffer and quantitated on an Agilent TapeStation 4200 System using the High Sensitivity D1000 ScreenTape.
Library strategy OTHER
Library source transcriptomic
Library selection other
Instrument model Illumina NextSeq 500
Description Single cell calling cards library
SRRs represent biological replicates.
Data processing Read 1 sequenced the cell barcode and unique molecular index of each self-reporting transcript. Read 2 began with GGTTAA (end of the piggyBac terminal repeat and insertion site motif) before continuing into the genome. Reads containing this exact hexamer were trimmed using cutadapt with the following settings: -g "^GGTTAA" --minimum-length 1 --discard-untrimmed -e 0 --no-indels. Reads passing this filter were then trimmed of any trailing P7 adapter sequence, again using cutadapt and with the following settings: -a "AGAGACTGGCAAGTACACGTCGCACTCACCATGANNNNNNNNNATCTCGTATGCCGTCTTCTGCTTG" --minimum-length 1. Reads passing these filters were aligned using 10x Genomics’ cellranger with the following settings: --expect-cells=6000 --nosecondary --chemistry=SC3Pv2 --localcores=16 --localmem=30. This workflow also managed barcode validation and collapsing of UMIs. Aligned reads were validated by verifying that they mapped adjacent to TTAA tetramers. Reads were then converted to calling card format (.ccf.txt). Finally, to minimize the presence of intermolecular artifacts, we required that each insertion must have been tagged by at least two different UMIs. We used the set of validated cell barcodes from each scRNA-seq library to demultiplex library-specific barcoded insertions from the scCC data. This approach requires no shared cell barcodes between individual scCC (and scRNA-seq) libraries. As a result, we excluded insertions from non-unique cell barcodes, which represented a very small number of total cells lost (< 1% per multiplexed library).
Genome_build: mm10
Supplementary_files_format_and_content: BAM files contain aligned and annotated calling card reads. Barcode files contain validated single cell barcodes. ccf.txt files contain processed calling card data. Peaks file provides details on regions significantly enriched for calling card insertions. describes calling card-specific BAM tags.
Submission date Apr 10, 2020
Last update date Jul 23, 2020
Contact name Robi D Mitra
Organization name Washington University in St. Louis
Department Genetics
Street address 4515 McKinley Ave
City St. Louis
State/province Missouri
ZIP/Postal code 63143
Country USA
Platform ID GPL19057
Series (1)
GSE148448 Self-reporting transposons enable simultaneous readout of gene expression and transcription factor binding in single cells
BioSample SAMN14573320
SRA SRX8094425

Supplementary file Size Download File type/resource
GSM4471660_Mouse-Cortex_Astrocyte_HyPBase_scCC_peaks.tsv.gz 10.9 Kb (ftp)(http) TSV
GSM4471660_Mouse-Cortex_Neuron_HyPBase_scCC_peaks.tsv.gz 26.8 Kb (ftp)(http) TSV
GSM4471660_Mouse-Cortex_Neuron_L2-4_HyPBase_scCC_peaks.tsv.gz 11.7 Kb (ftp)(http) TSV
GSM4471660_Mouse-Cortex_Neuron_L2-4_v_L5-6_HyPBase_scCC_peaks.tsv.gz 797 b (ftp)(http) TSV
GSM4471660_Mouse-Cortex_Neuron_L5-6_HyPBase_scCC_peaks.tsv.gz 10.3 Kb (ftp)(http) TSV
GSM4471660_Mouse-Cortex_Neuron_L5-6_v_L2-4_HyPBase_scCC_peaks.tsv.gz 729 b (ftp)(http) TSV
GSM4471660_Mouse-Cortex_Neuron_downsample_HyPBase_scCC_peaks.tsv.gz 5.7 Kb (ftp)(http) TSV
GSM4471660_Mouse-Cortex_combined_scCC.bam 385.2 Mb (ftp)(http) BAM
GSM4471660_Mouse-Cortex_combined_scCC.ccf.txt.gz 1.5 Mb (ftp)(http) TXT
GSM4471660_Mouse-Cortex_combined_scCC_barcodes.csv.gz 213.4 Kb (ftp)(http) CSV
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

| NLM | NIH | GEO Help | Disclaimer | Accessibility |
NCBI Home NCBI Search NCBI SiteMap