Single-cell transcriptome profiling via Smart-seq2
Whole transcriptome amplification, library preparation and sequencing of single cells/nuclei were performed using the Smart-seq2 modified protocol21 (link),33 (link),35 (link),68 (link),69 (link). RNA was purified with Agencourt RNAClean XP beads (Beckman Coulter). Oligo-dT primed reverse transcription (RT) was performed using Maxima H Minus reverse transcriptase (Life Technologies) and a template-switching oligonucleotide (TSO; Qiagen). PCR amplification (20 cycles for scRNA-seq and 22 cycles for snRNA-seq) was performed using KAPA HiFi HotStart ReadyMix (KAPA Biosystems), followed by Agencourt AMPure XP bead (Beckman Coulter) purification. Libraries were generated using the Nextera XT Library Prep kit (Illumina). Libraries from 768 cells with unique barcodes were combined and sequenced using a NextSeq 500/550 High Output Kit v2.5 (Illumina).
Liu I., Jiang L., Samuelsson E.R., Marco Salas S., Beck A., Hack O.A., Jeong D., Shaw M.L., Englinger B., LaBelle J., Mire H.M., Madlener S., Mayr L., Quezada M.A., Trissal M., Panditharatna E., Ernst K.J., Vogelzang J., Gatesman T.A., Halbert M.E., Palova H., Pokorna P., Sterba J., Slaby O., Geyeregger R., Diaz A., Findlay I.J., Dun M.D., Resnick A., Suvà M.L., Jones D.T., Agnihotri S., Svedlund J., Koschmann C., Haberler C., Czech T., Slavc I., Cotter J.A., Ligon K.L., Alexandrescu S., Yung W.K., Arrillaga-Romany I., Gojo J., Monje M., Nilsson M, & Filbin M.G. (2022). The landscape of tumor cell states and spatial organization in H3-K27M mutant diffuse midline glioma across age and location. Nature Genetics, 54(12), 1881-1894.
Corresponding Organization : Dana-Farber/Boston Children's Cancer and Blood Disorders Center
Other organizations :
Science for Life Laboratory, Stockholm University, Ludwig-Maximilians-Universität München, Broad Institute, Comprehensive Cancer Center Vienna, Medical University of Vienna, Stanford University, Hopp Children's Cancer Center Heidelberg, German Cancer Research Center, Heidelberg University, Dana-Farber Cancer Institute, University of Pittsburgh, Neurological Surgery, Central European Institute of Technology, Central European Institute of Technology – Masaryk University, Masaryk University, University Hospital Brno, University of California, San Francisco, University of Newcastle Australia, Children's Hospital of Philadelphia, Michigan Medicine, Children's Hospital of Los Angeles, University of Southern California, Boston Children's Hospital, The University of Texas MD Anderson Cancer Center, Massachusetts General Hospital
Whole transcriptome amplification, library preparation and sequencing of single cells/nuclei
dependent variables
RNA expression
control variables
Agencourt RNAClean XP beads (Beckman Coulter) for RNA purification
Maxima H Minus reverse transcriptase (Life Technologies) and a template-switching oligonucleotide (TSO; Qiagen) for oligo-dT primed reverse transcription (RT)
KAPA HiFi HotStart ReadyMix (KAPA Biosystems) for PCR amplification
Agencourt AMPure XP beads (Beckman Coulter) for purification
Nextera XT Library Prep kit (Illumina) for library generation
NextSeq 500/550 High Output Kit v2.5 (Illumina) for sequencing
controls
No positive or negative controls were explicitly mentioned in the protocol.
Annotations
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