title: IC21
organism: Rattus norvegicus
description: Protocols: NS1 is a GFP positive tumor cell line which can be syngeneically inoculated into Fischer 344 rats where it generates infiltrative tumors, with perivascular dissemination. The rat glioma cells (NS1) were cultured using RPMI-1640 (Sigma-Aldrich) medium with addition of 1% ml Na-pyruvate, 1% ml HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), 0.1% ml Gentamycin, as well as 10% inactivated fetal calf serum (heated to 56 °C for 30 minutes). NS1 cells were prepared for inoculation by removal of the medium and washed gently with PBS as described above. 5 000 cells were used for intracranial inoculations. Intracerebral inoculation was done under isoflurane inhalation anaesthesia using a stereotactic frame and a 10 µl Hamilton syringe. The cells were injected at a depth of 5 mm from the skull, 2 mm laterally from the sagittal suture, and 1 mm anterior to the coronal suture, on the right side of the cranium. The cranial burr hole was sealed with bone wax, and the incision was closed with absorbable suture. Animals were monitored on a daily basis and euthanized if they met the criteria defined by the ethics regulations, including impaired general condition and neurological deficits. In animals that were euthanatized due to intracranial tumor growth, part of the tumor was dissected and frozen in liquid nitrogen. Animals without any visible tumor growth at the end of the observation period were thus not candidates for RNA extraction. The rat glioma cells (NS1) that were used to generate intracranial tumors were cultured using RPMI-1640 (Sigma-Aldrich) medium with addition of 1% ml Na-pyruvate, 1% ml HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), 0.1% ml Gentamycin, as well as 10% inactivated fetal calf serum (heated to 56 °C for 30 minutes), as previously described. Immune therapy was delivered with intratumoral administration of 0.4 ul anti-C1-IA (6.15 mg/ml) (Covance) day 0 and 10. Radiotherapy was delivered at 8 Gy x 2 day 8 and 12 for intracranial tumors, at a 220 kV preclinical research platform (XenX, XStrahl Inc, Suwanee, GA, USA). RNA extraction was performed using RNeasy kit (Qiagen) according to the manufacturer's instructions 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)