Merge pull request #39 from mkoptyra/master

CBTTC: specimens pipeline, cell lines generation, DNA/RNA extraction and data generation section of materials and methods

content/03.methods.md

@@ -3,20 +3,50 @@
 ### Biospecimen collection
 
 The Pediatric Brain Tumor Atlas specimens are comprised of samples from Children's Brain Tumor Tissue Consortium (CBTTC) and the Pediatric Pacific Neuro-oncology Consortium (PNOC). 
-Blood and tumor biospecimens from patients enrolled within the CBTTC were sent to the Children's Hospital of Philadelphia for RNA and/or DNA extraction.
+
+#### Children's Brain Tumor Tissue Consortium (CBTTC)
+
+The CBTTC [@url:https://cbttc.org] is a collaborative, multi-institutional (16 institutions worldwide) research program dedicated to the study of childhood brain tumors. 
+All CBTTC data can be download from the Gabriella Miller Kids First Data Resource Center (KF-DRC), [@url:https://kidsfirstdrc.org].
+The deidentified patient's blood and tumor tissue were prospectively collected by the consortium from patients enrolled within the CBTTC. 
+
+The cell lines were generated by the CBTTC from either fresh tumor tissue obtained firectly from surgery performed at Children’s Hospital of Philadelphia (CHOP) or from prospectively collected tumor specimens stored in Recover Cell Culture Freezing media (cat# 12648010, Gibco).
+The tissue was dissociated using enzymatic method with papain as described [@doi:10.1101/656587].
+Briefly, tissue was washed with HBSS (cat# 14175095, Gibco), minced and icubated with activated papin solution (cat# LS003124, SciQuest) for up to 45 minutes. 
+The papain was inactivated using ovomucoid solution (cat# 542000, SciQuest), tissue was briefly treated with DNase (cat#  10104159001, Sigma) and passed through the 100μm cell strainer (cat# 542000, Greiner Bio-One). 
+Two cell culture conditions were initiated based on the number of cells avialble. 
+For cultures utilizing the fetal bovine serum (FBS), a minimum density of 3×10^5 cells/ml were plated in DMEM/F-12 medium (cat# D8062, Sigma) supplemented with 20% FBS (cat# SH30910.03, Hyclone), 1% GlutaMAX (cat# 35050061, Gibco), Penicillin/Streptomycin-Amphotericin B Mixture (cat# 17-745E, Lonza) and 0.2% Normocin (cat# ant-nr-2, Invivogen). 
+For the serum-free media conditions cells were plated at minimum density of 1×10^6 cells/ml in DMEM/F12 media supplemented with 1% GlutaMAX, 1x B-27 supplement minus vitamin A (cat# 12587-010, Gibco), 1x N-2 supplement (cat# 17502001, Gibco), 20 ng/ml epidermal growth factor (cat# PHG0311L, Gibco), 20 ng/ml basic fibroblast growth factor (cat# 100-18B, PeproTech), 2.5μg/ml heparin (cat# H3149, Sigma), Penicillin/Streptomycin-Amphotericin B Mixture and 0.2% Normocin.
+
+#### Pacific Pediatric Neuro Onclology Consortium (PNOC)
+
+The Pacific Pediatric Neuro-Oncology Consortium (PNOC) is an international consortium dedicated to bringing new therapies to children and young adults with brain tumors.
 PNOC collected blood and tumor biospecimens from newly-diagnosed DIPG patients as part of the clinical trial [PNOC003/NCT02274987](https://clinicaltrials.gov/ct2/show/NCT02274987) [@doi:10.1002/ijc.32258]. 
 
-#### Nucleic acids extraction and library preparation
+### Nucleic acids extraction and library preparation
 
-##### PNOC samples
+#### PNOC samples
 The Translational Genomic Research Institute (TGEN; Phoenix, AZ) performed DNA and RNA extractions on tumor biopsies using a DNA/RNA AllPrep Kit (Qiagen, #80204). 
 All RNA used for library prep had a minimum RIN of 7 but no QC thresholds were implemented for the DNA.
 For library preparation, 500ng of nucleic acids were used as input for RNA-Seq and WXS.
 The RNA prep was performed using the TruSeq RNA Sample Prep Kit (Illumina, #FC-122-1001) and the exome prep was performed using KAPA Library Preparation Kit (Kapa Biosystems, #KK8201) using Agilent's SureSelect Human All Exon V5 backbone with custom probes. 
 These probes include CGH probes that target 44,000 evenly spaced genomic loci to assess copy number changes, as well as probes that tile across tumor suppressor genes and genes involved in common cancer translocations.
 All extractions and library preparations were performed according to manufacturer's instructions.
 
-##### CBTTC samples
+#### CBTTC samples
+
+Blood, tissue and cell line DNA/RNA extraction was performed at Biorepository Core (BioRC) at CHOP. 
+Briefly, 10-20 mg frozen tissue, 0.4-1ml of blood or 2×10^6 cells pellet was used for extractions.
+Tissues were lysed using a Qiagen TissueLyser II (Qiagen) with 2×30 sec at 18Hz settings using 5 mm steel beads (cat# 69989, Qiagen). 
+Both tissue and cell pellets processes included a CHCl3 extraction and were run on the QiaCube automated platform (Qiagen) using the AllPrep DNA/RNA/miRNA Universal kit (cat# 80224, Qiagen). 
+Blood was thawed and threated with RNase A (cat#, 19101, Qiagen); 0.4-1ml was processed using the Qiagen QIAsymphony automated platform (Qiagen) using the QIAsymphony DSP DNA Midi Kit (cat# 937255, Qiagen).
+DNA and RNA quantity and quality was assessed by PerkinElmer DropletQuant UV-VIS spectrophotometer (PerkinElmer) and an Agilent 4200 TapeStation (Agilent, USA) for RINe and DINe (RNA Integrity Number equivalent and DNA Integrity Number equivalent respectively). 
+Library preparation and sequencing was performed by the NantHealth sequencing center. 
+Briefly, DNA sequencing libraries were prepared for tumor and matched-normal DNA using the KAPA Hyper prep kit (cat# KK8541, Roche); tumor RNA-Seq libraries were prepared using KAPA Stranded RNA-Seq with RiboErase kit (cat# KK8484, Roche). 
+Whole genome sequencing (WGS) was performed at an average depth of coverage of 60X for tumor samples and 30X for germline. 
+RNA samples were sequenced to an average of 200M reads. 
+All samples were sequenced on the Illumina HiSeq platform (X/400) (Illumina) with 2 × 150bp read length.
+For the cell line sequencing, samples labelled with “CL-adh” correspond to the adherent FBS cell lines and those labelled “CL-susp” are the serum-free lines.
 
 #### Data generation