- Preparing Samples for Submission
After harvesting cells, do one of the following:
1. Pellet cells, snap freeze in liquid nitrogen and store at -90C to -65C. Deliver samples on dry ice. 🧊
2. Pellet cells, remove supernatant, store dry cell pellet at -80C. Deliver samples on dry ice. 🧊
3. Lyse cells in Buffer RLT (Contact HSSC for Buffer information). Deliver samples on wet ice. 💧
Tissue/ Paraffin-embedded Tissue (FFPE)
Deliver tissue on dry ice 🧊
Deliver at room temperature.
Blood, Serum, Plasma, Buffy Coat
Frozen samples: deliver on dry ice
Not frozen: deliver on wet ice 💧
NGS Samples for Library Preparation
Dry ice 🧊
Wet ice 💧
Wet ice 💧 or directly in freezer 🏥
Wet ice 💧 or directly in freezer 🏥
- Before making your libraries, please make sure your barcodes are compatible with Illumina Standards
- Minimum concentration: 2 nM (measured by fluorometry)
- Minimum volume: 15-20 μl
Single Cell Core
- Single cell samples should be brought to us as a cell suspension in PBS containing 0.04% BSA.
Nuclei for Multiome processing should be brought in Nuclei Resuspension Buffer with RNAse Inhibitor.
Fixed Cells for the Flex Assay should be prepared in long term storage buffer and delivered on dry ice.
- Cells should be as concentrated as possible. Samples run individually should be ~1 million live cells per mL. Samples intended for cell hashing should be ~3.5 million live cells per mL. Nuclei for Multiome processing should be 3.5 million nuclei per mL.
- Cell loading recommendations are 500-10,000 cells per well or up to 30,000 cells per well for hashed samples. The doublet rate and chances of clogging the chip increase with higher cell loading.
- If your sample dissociation protocol contains Tween-20 or EDTA, you will need to ensure that this is washed out of your sample prior to submission as this can prevent emulsification.
- Cells should be filtered through at least a 70 micron filter (30-40 micron recommended). Cells should be smaller than 54 microns (the channel size of the Chromium chip).
- Drop-off Locations and Instructions
- Samples must be labeled with PI name, contact name, email and phone number.
- Call (412-692-3555) or email us within 2 hours to let us know you have dropped off samples so we may retrieve them.
- Send sample submission form with all information. Samples cannot be processed without a proper sample submission form.
We are located in the Rangos Research Bldg, on the Children’s Hospital Campus in Lawrenceville. There is a drop-off freezer just inside the sliding doors at the entrance to our building.
If you are driving, you can pull into the driveway for the Mid-Campus garage on 44th St. and follow past the Plaza Building/Ronald McDonald House to the front entrance to drop-off. There is a small area to turn around in or you can park in the Mid-campus garage (pay-parking for visitors and patient families). Once you park, go to the ground floor, walk through the garage towards Rangos (instead of towards the main hospital), and you will come to the ground floor Rangos entrance.
Single Cell Core
The single cell core is located in S765 in the South BST. Our lab phone number is 412-383-5223.
- Research Support
Health Sciences Sequencing Core
Facilities and Other Resources description for Grant applications:
The Health Sciences Sequencing Core (HSSC) at Children’s Hospital of Pittsburgh is housed in the Richard King Mellon Institute for Pediatric Research, which is located on the 8th floor of the new Rangos Research Building as part of the Children’s Hospital of Pittsburgh of UPMC. The RK Mellon Institute for Pediatric Research is a multidisciplinary research institute comprising of physician scientists in the fields of Immunology, Developmental Biology, and Neurology. The John G. Rangos Research Center is a nine story, 144,000 square foot building located on the newly constructed, environmentally “green” campus of Children’s Hospital of Pittsburgh of UPMC. The total area of the RK Mellon Institute for Pediatric Research laboratories and facilities is around 10,000 square feet. Our facility represents around 500 square feet and offers next generation sequencing services to all University of Pittsburgh Researchers.
Next Generation Sequencing: The facility operates Illumina NextSeq 2000 & 500 instruments and an Illumina DRAGEN server on the NextSeq 2000 that can generate up to 330Gb of sequence data per flow cell. Instrument capacity is therefore readily available for large scale sequencing projects using sequencing by synthesis technology. Instruments can be operated in medium or high output to facilitate projects of any scale. In addition, the facility operates an Illumina MiSeq instrument for targeted resequencing, small genome, metagenomic and microbiome studies, as well as QC assessment. Together these platforms provide for a broad range of applications from targeted resequencing to genomic scale sequencing capability. A Fragment Analyzer (Advanced Analytical Inc.), a Qubit Fluorometer (Thermo Fisher) and a 4150 TapeStation (Agilent) are used for library QC and quantification. For Spatial Transcriptomics, the HSSC has a dedicated EVOS M7000 Imaging System equipped with 4x, 10x, 20x objectives, and GFP, DAPI, CY5, RFP light cubes for IF imaging workflows.
Facility dedicated equipment includes: one illumina NextSeq 2000 high throughput sequencer, one illumina NextSeq 500 high throughput sequencer, one illumina MiSeq sequencer, three Forma Freezers (-80°C; Upright); two Forma Freezers (-20°C; Upright); Agilent 4150 TapeSation; Advanced Analytical Inc. 5300 Fragment Analyzer; Invitrogen Qubit 4.0; Invitrogen Qubit FLEX; NanoDrop 2000 UV-Vis Spectrophotometer; Bio-Rad CFX96 Touch Real-Time PCR Detection System; two C1000 Touch Thermal Cyclers, two T100 Touch Thermal Cyclers, Liquid Nitrogen Storage Container DOT Scientific and multiple high-speed and ultra-centrifuges, EVOS M7000 Imaging System
Please acknowledge work from our core in all publications by including the following statement: This project utilized the services of the University of Pittsburgh Health Sciences Sequencing Core at UPMC Children’s Hospital of Pittsburgh, for [name(s) of relevant service(s)].
- Sample Submission Forms