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==February 10, 2015 bulk nextera== Motivation: I want to make bulk libraries from 1-02 (AD) and 25-01 (CTL) using SM59 with our new IDT-Nextera v2 duel index primers, but I first need to test SM59 against 1:50 Nextera v1 with single index primers. Therefore, my plan is to first extract DNA samples from the tubes I have in excess (cerebellum) to compare SM59 to Nextera v1, and wait on the more precious tubes (cortex). Brandon already tested dilutions of SM59 and EZ-Tn5 (N = 1 per concentration) on HLS lysed cells (for accessibility), but it seems that the variability in lysis might be masking the dilution effect (no clear trend). He did include 6 ng purified genomic, which gave a very bright smear. I will use HLS-GHCl lysis followed by Ampure XP, pick one concentration of SM59 and Nextera v1 with more replicates. Goal: First, to extract, purify, and freeze six 1000 nuclei bulk DNA samples from 1-02 cerebellum [http://genome-tech.ucsd.edu/LabNotes/index.php/Arichard:Samples/sort141009 sorted October 9, 2014]. Second, create three libraries each with 1X SM59 (Stock is 5X) and 1:50 Nextera v1 and compare gels for intensity and distribution. Third, use SM59 to generate bulk libraries from 1-02 and 25-01 cortex with N = 3 for all. ===Samples=== To test SM59 {| {{table}} | align="center" style="background:#f0f0f0;"|'''sort''' | align="center" style="background:#f0f0f0;"|'''# on tube''' | align="center" style="background:#f0f0f0;"|'''nuclei/ul''' | align="center" style="background:#f0f0f0;"|'''ul/tube''' | align="center" style="background:#f0f0f0;"|'''nuclei/sample''' | align="center" style="background:#f0f0f0;"|'''# samples''' | align="center" style="background:#f0f0f0;"|'''# tubes to pool''' | align="center" style="background:#f0f0f0;"|'''ul/sample''' | align="center" style="background:#f0f0f0;"|'''ul HLS''' |- | 1-02 cbl full||4||274||10||1000||6||3||3.65||31.35 |- | |} Use 8-tube strip, including positive and negative control Remaining bulk libraries {| {{table}} | align="center" style="background:#f0f0f0;"|'''sort''' | align="center" style="background:#f0f0f0;"|'''# on tube''' | align="center" style="background:#f0f0f0;"|'''nuclei/ul''' | align="center" style="background:#f0f0f0;"|'''ul/tube''' | align="center" style="background:#f0f0f0;"|'''nuclei/sample''' | align="center" style="background:#f0f0f0;"|'''# samples''' | align="center" style="background:#f0f0f0;"|'''# tubes to pool''' | align="center" style="background:#f0f0f0;"|'''ul/sample''' | align="center" style="background:#f0f0f0;"|'''ul HLS''' |- | 1-02 ctx full||1||120||3||1000||3||Not enough||NA||NA |- | 1-02 ctx hi||2||120||3||1000||3||Not enough||NA||NA |- | 1-02 ctx lo||3||78||10||1000||3||4||12.82||22.18 |- | 25-01 ctx full||5||283||20||1000||3||1||3.53||31.47 |- | 25-01 cbl full||6||232||10||1000||3||2||4.31||30.69 |- | |} ===Protocol=== Following protocol from [http://genome-tech.ucsd.edu/LabNotes/index.php/Arichard:Notebook/bulk150122#Bulk_libraries_January_22.2C_2015a January 22] up to Nextera ====Lysis and freezedown==== # Lyse in 35 ul [http://genome-tech.ucsd.edu/LabNotes/index.php/Arichard:Reagents/hls 1X HLS] at RT for <5 min, followed by 35 ul of 8M guanidine HCl. # 126 ul (1.8:1 ratio) Ampure XP, washed 2X with 80% EtOH # Elute in 7 ul H2O # Freeze at -20 C ====Tagmentation==== {| {{table}} | align="center" style="background:#f0f0f0;"|'''Tube''' | align="center" style="background:#f0f0f0;"|'''1''' | align="center" style="background:#f0f0f0;"|'''2''' | align="center" style="background:#f0f0f0;"|'''3''' | align="center" style="background:#f0f0f0;"|'''4''' | align="center" style="background:#f0f0f0;"|'''5''' | align="center" style="background:#f0f0f0;"|'''6''' | align="center" style="background:#f0f0f0;"|'''7''' | align="center" style="background:#f0f0f0;"|'''8''' |- | Sample||1-02 cbl||1-02 cbl||1-02 cbl||1-02 cbl||1-02 cbl||1-02 cbl||NTC||6 ng gDNA |- | Condition||1:50 Nextera v1||1:50 Nextera v1||1:50 Nextera v1||1X SM59||1X SM59||1X SM59||1X SM59||1X SM59 |- | |} * Using SM59 on 5 tubes: 1-02 cerebellum 4-6, positive control, and NTC with supplied buffer. ** Must mix A and B 1:1 --> 3 ul each * Using 1:50 Nextera v1 on 1-02 cerebellum 1-3 with in-house HMW buffer. * 5 minutes at 55 C * Standard ProtQ and Exo- treatments after tagmentation ====PCR==== * Normal 2 step (6+7) PCR with indices 1, 2, and 5 for Nextera v1 tubes * 13 cycles with N5xx and N7xx for SM59 tubes
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