title: Long-term anti-tumor effects following both conventional radiotherapy and FLASH in fully immunocompetent animals with glioblastoma
design: Long-term anti-tumor effects following both conventional radiotherapy and FLASH in fully immunocompetent animals with glioblastoma
library_construction_protocol: Rats are 10 weeks old, female Fisher 344 rats at the initiation of the experiments. Tumor cell line: NS1 cells -  can be syngeneically inoculated into Fischer 344 rats where it generates infiltrative tumors, described by Ahlstedt, J., et al., Growth pattern of experimental glioblastoma. Histol Histopathol, 2020. 35(8): p. 871-886.Intracranial tumor: Each rat received 5000 NS1 glioblastoma cells, suspended in 5 µl of serum free nutrient solution. A stereotactic frame and a 10 µl Hamilton syringe. Isofluran anaesthesia was applied. The cells were injected on the right side of the cranium at a depth of 5 mm, 2 mm laterally from the sagittal suture, and 1 mm anterior to the coronal suture. The cranial burr hole was sealed with bone wax, and the incision was closed with absorbable suture.Subcutaneous tumor: Rats were inoculated with 50 000 NS1 cells subcutaneously on their hindleg and tumor site was carefully marked.  Upon tumor development, a part of the tumor was dissected and frozen in liquid nitrogen when the. RNA extraction was performed using RNeasy kit (Qiagen®) according to the manufacturer's instructions.  All experiments have been approved by the local ethics board and have been conducted in accordance with the regulations. Tumors were collected from rats with intracranial or subcutaneous glioma. RNA was extracted using RNeasy kit (Qiagen®) according to manufacturer's instructions. a. Protocol: Qubit Flex Fluorometer Quick Reference No. MAN0018187 Rev. A.0 b. Reagent/kit: QuantIT RNA HS Assay Kit (Q33140, Life Technologies) c. Equipment: Qubit Flex (Q33327, Invitrogen Thermo Fisher Scientific) III. Library preparation: a. Protocol: TruSeq® Stranded mRNA Sample Preparation Guide (Part # 15031047 Rev. E) Modification of “Enrich DNA Fragment” step: from 15 to 13 PCR cycles b. Reagent/kit: TruSeq® Stranded mRNA Library Prep (20020594, Illumina) c. Index: IDT for Illumina TruSeq RNA UD Indexes (96 indexes, 20022371, Illumina) d. Equipment: Automatisation of Clean up steps: King Fisher FLEX (18-5400620, Thermo Scientific), incubations and PCR: Eppendorf Mastercycler X50s (6311000010, Eppendorf)  IV. Library concentration check a. Protocol: Qubit Flex Fluorometer Quick Reference No. MAN0018187 Rev. A.0 b. Reagent/kit: QuantIT 1X dsDNA HS Assay Kit (Q33232) c. Equipment: Qubit Flex (Q33327 Invitrogen Thermo Fisher Scientific)  V. Library size and quality check a. Protocol: Agilent D5000 ScreenTape Quick Guide for TapeStation, Part Number: G2991-90150 Edition 08/2015 b. Reagent/kit: D5000 Screen Tape (5067-5588, Agilent) c. Equipment: Agilent 2200 TapeStation System (G2964AA, Agilent)