uote what I should annotate and for each annotation give me a one sentence comment I should say about the highlighted portion : Look over the slides from Week 1 "How to read a scientific article," and consider addressing these topics: 1) The major research question(s) explored. 2) What prior research was done? What research gaps does the author intend to fulfill? 3) What are their hypotheses? 4) How does the author address these gaps (i.e., methods)? 5) What's the main finding of each figure? 6) If you can choose, what is the ONE piece of data that was most important or directly addressing the question? 7) Do the author's interpretations match the evidence? (It's OK to disagree!) 8) Any critiques or further comments on the paper.

Semi-quantitative real-time RT-PCR Ribonucleic acid from FACS-sorted cells was isolated using the Arcturus PicoPure RNA Isolation Kit (Applied Biosystems, Waltham, MA, United States, #KIT0204) according to the manufacturer's protocol. Sorted 04+ cells were resuspended in 100 ul RNA extraction buffer and incubated at 42C for 30 min. Samples were spun down and the RNA-containing supernatant further purified of residual genomic DNA by on-column digestion with RNase-free DNase (Qiagen, Germantown, MD, United States, #79254). Total RNA (5 ug) was reverse transcribed with Sensiscript RT Kit (Qiagen, Germantown, MD, United States, #205211) with random primers according to manufacturer's protocol. Resulting cDNA was preamplified in 20 ul reactions using PowerUp SYBR Green Master Mix (Applied Biosystems, Waltham, MA, United States, #A25742), with 40 nM of each PCR primer and 4 ul of template cDNA. The following thermal profile was used: Initial denaturation: 95 C, 3 min; Cycle (20 cycles total): Denaturation, 95C, 20 sec; Amplification, 57C, 3 min; Extension: 72 C, 1 min; Final extension: 72 C, 10 min. Primers were checked for specificity and correct product length by regular RT-PCR followed by agarose gel electrophoresis. Semi quantitative RT- PCR was performed using the PowerUp SYBR Green Master Mix (Applied Biosystems, Waltham, MA, United States) and preamplified cDNA as a template with the following thermal profile: 50 C for 2 min; 95C for 2 min; 40 cycles of 95 C for 15 s, 57 C for 20 s, 72 C for 1 min; Final step: 95 C for 15 s, 57C for 1 min, and 95 C for 15 s. All samples were run in duplicate in the QuantStudio 6 Flex Real Time PCR system (Applied Biosystems, Waltham, MA, United States). Relative gene expression was calculated with the AACt method after normalization to glyceraldehyde-3-phosphate de-hydrogenase (GAPDH) as reference gene