title: GSM9210468: White blood cells (CapSeq with and without hairpin ligation); Homo sapiens; RNA-Seq
library_construction_protocol: White blood cells were prepared by removing red blood cells (RBC) in the following manner. Peripheral blood was collected within the last 2 hours into a K2EDTA vacutainer and stored on wet ice. Subsequently, 10 µL of 0.5 M EDTA (Invitrogen, cat. no. 15575-038) was added per 1 mL of peripheral blood. In 2 mL Protein LoBind tube (Eppendorf, cat. no. 0030108132) 1800 µL of RBC lysis buffer (R7757, Sigma-Aldrich) was combined with 200 µL of peripheral blood. Cells with RBC lysis buffer were incubated for 5 min at room temperature and the tube was gently inverted every 5 s. Next, cells were centrifuged for 10 min at 300g and 4 °C using a swinging-bucket rotor. After discarding the supernatant, the pellet was gently resuspended with 2 mL RBC lysis buffer. The tube was incubated for 3 min at room temperature and gently inverted every 5 s. Then the cells were centrifuged for 5 min at 300g and 4 °C using a swinging-bucket rotor, and supernatant was discarded. Additional wash in RBC lysis buffer was performed if very bright red color was still visible on the pellet. Finally, the pellet was resuspended to the final volume of 100 µL with ice cold 1X DPBS. Cells were fixed by slowly adding 900 µL of ice-cold methanol into the 100 µL of cell suspension in 2 mL Protein LoBind tube. To avoid cell clumping the first 500 µL of the methanol was added drop by drop (3-5 drops per 1 s) and the remaining volume was added more quickly (in 1-2 s). After methanol was added, the tube was gently inverted 10-times. Next, the tube with methanol-fixed cells was incubated on ice for 15-30 min and transferred at -20 °C (for short period <6 months) or at -80 °C (for longer period >6 months). The fixed cells from the freezer were initially incubated on ice for 15-30 min followed by centrifugation for 5 min at 1000g and 4 °C using a swinging-bucket rotor. Next, the methanol was removed, and the cell pellet was resuspended in 1 mL of rehydration buffer (RB; 1X SSC, 2 M NaCl, 0.1% BSA) and transferred to a new 1.5 mL tube. The resuspended cells were then subjected to an additional centrifugation step for 5 min at 1000g and 4 °C. Subsequently, the supernatant was discarded, and the cells were subjected to an identical wash procedure using 1 mL of RB. A 10 µL aliquot of the cell suspension was utilized to determine the cell concentration using a hemocytometer. Following removal of the supernatant, the cell pellet was resuspended to achieve a final volume corresponding to 3 million cells per milliliter. Illumina sequencing libraries were prepared for each amplified cDNA aliquot with NEBNext® Ultra™ II FS DNA Library Prep Kit for Illumina (NEB, cat. no. E7805S) according to the instructions in the manual, with minor changes. In particular, 5-10 ng of amplified cDNA was used for cDNA fragmentation and transferred to a PCR tube. Nuclease-free water was added to reach the final volume on 13 µL. Fragmentation reaction was assembled on ice by adding 3.5 µL of NEBNext® Ultra™ II FS Reaction Buffer followed by 1 µL of NEBNext® Ultra™ II FS Enzyme Mix. Reaction mix was vortexed and spun-down for 3-5 seconds. The fragmentation was performed for 8 minutes at 37 °C, followed by end-repair and dA tailing for 30 min at 65 °C (lid was set at 75 °C). Fragmented cDNA was purified with 0.6X-0.8X double-size selection using SPRIselect beads and eluted in 17.5 µL with nuclease-free water. Then, 1.5 µM ligation adapter was prepared in advance by mixing 1.5 µL of forward ligation primer (100 µM, /5Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCAC/3ddC; HPLC purification, IDT) and 1.5 µL of reverse ligation primer (/5AmMC6/GCTCTTCCGATCT; HPLC purification, IDT) with 97 µL of water. To anneal oligos, the solution was heated to 95 °C for 2 min (lid at 100 °C) and cooled at room temperature for 5 min. Next, purified fragmented cDNA was mixed with 15 µL of NEBNext® Ultra™ II Ligation Master Mix, 1.25 µL of ligation adapter and 0.5 µL of NEBNext® Ligation Enhancer. Reaction solution was well mixed by pipetting and incubated for 15 min at 20 °C (lid set at 37 °C). The ligation product was diluted to 100 µL with nuclease-free water, followed by a 0.8X SPRI size selection procedure and eluted in 40 µL with nuclease-free water. Finally, the ligation product was amplified by 12-cyles of PCR: 95 ºC for 3 min, 12-cycles [98 ºC for 20 s, 54 ºC for 30 s, 72 ºC for 20 s], and 72 ºC for 60 s. PCR mix was prepared by mixing 20 µL of ligation product, 2.5 µL of forward indexing primer (Table  S25), 2.5 µL of reverse indexing primer (Table S25) and 25 µL of 2X KAPA HiFi HotStart ReadyMix. Amplified sequencing libraries were purified with 0.6X-0.8X double-size selection using SPRIselect beads and eluted in 20 µL with nuclease-free water.