title: GSM4668815: Healthy donor, normal density neutrophils (NDN), replicate 3; Homo sapiens; RNA-Seq
library_construction_protocol: RNA was extracted using Qiagen's RNAeasy Plus Microkit. Library Preparation: Libraries were prepared using the Universal Plus mRNA-Seq with (NuGEN Cat# 0520-24).  a. Poly(A) Selection: 8-30 ng of RNA samples (in a volume of 50 μl) were mixed with  Oligo (dT) Beads and incubated at 65oC for 5 minutes, mixed, then at room  temperature for 10 mins. The supernatant was discarded and the beads were  washed with wash buffer. Captured polyadenylated RNA was eluted using Elution  buffer at 80°C for 2 min. mRNA is further purified in a second bead clean-up.  b. RNA Fragmentation: Beads were mixed with 20 μl Fragmentation Buffer and  incubated for 8 minutes at 94oC. 20 μl of supernatant was removed from the beads  and proceeded immediately to First Strand cDNA Synthesis.  c. First Strand cDNA Synthesis: 5 μl of First Strand Master Mix was added to each  sample and placed on a thermal cycler to run preprogrammed thermal conditions.  d. Second Strand cDNA Synthesis: 50 μl Second Strand Master Mix was added to  each sample, mixed well and incubated in a thermal cycler at 16oC for one hour. e. cDNA Purification: cDNA was purified using Agencourt AMPure XP Beads (1.8  volumes), eluted in 10 μl of H2O, and stored at -20oC.  f. End Repair: End Repair Mix was added to the purified samples from step e and the  samples were incubated in a preprogrammed thermal cycler.  g. Adaptor Ligation: Samples were mixed with 3 μl adaptor mix, then 12 μl Ligation  Mix, and incubated in a thermal cycler at 25oC for 30 minutes.  Sample and Barcode Information: Samples were barcoded with NuGEN Unique  Dual Indexes. Strand Selection: 70 μl of Strand Selection Master Mix was added to 30 μl of each  sample, incubated in a pre-heated thermal cycler at 72oC for 10 minutes. Strand Selection Purification: Strand selected cDNA was purified using  Agencourt AMPure XP Beads (0.8 volumes), eluted in 15 μl of H2O, and stored at -  20oC.  j. Library Amplification: 76.5 μl of Library Amplification Master Mix was added to  13.5 μl of each sample and incubated in a preprogrammed thermal cycler, for 13-  16 cycles, which was pre-optimized with qPCR.  k. Amplified Library Purification: Amplified libraries were purified using 90 μl  Agencourt AMPure XP Beads (1.0 volumes). 25 μl of eluted libraries were collected  and stored at -20oC.  l. Validate Library: The concentration of libraries were measured by Qubit dsDNA  HS Assay Kit (Invitrogen Q32851). Libraries were diluted and normalized to the  optimal range for Agilent Bioanalyzer analysis using the DNA High Sensitivity Kit  (Agilent Technologies, Cat# 5067-4626).  m. Normalize and Pool Libraries: Same amount of libraries were pooled based on  the molar concentration from Bioanalyzer measurements. Library Denaturing and Diluting for MiSeq Nano 300: Pooled library was run on MiSeq  to test quantity and quality, using the MiSeq Reagent Nano Kit V2 300 cycles (Illumina, Cat.  No. MS-103-1001). Library and PhiX control (Illumina, Cat. No. FC-110-3001) were  denatured and diluted using the standard normalization method following manufacturer's  directions, to a final concentration of 6pM. Equal volume of library and PhiX were combined and  sequenced on Illumina MiSeq.  (https://basespace.illumina.com/run/196126950/JY_Xiaoling07072020_test)  3. Library Denaturing and Diluting for Nextseq 500: Library and PhiX were denatured and  diluted using the standard normalization method following manufacturer's directions. The  loading of library pool was 1.3 ml at 1.8 pM, with 1% PhIX spike in.  Sequencing Run: Sequencing was performed on the University of Louisville Brown Cancer  Center Genomics Core Illumina NextSeq 500 using the NextSeq 500/550 75 cycle High  Output Kit v2.5 (20024906).
attributes.tag: GEO Accession
attributes.value: GSM4668815